Recombinant adeno-associated vector-mediated delivery of B-domain-deleted factor VIII constructs for the treatment of hemophilia

ABSTRACT

One form of a composition has two types of recombinant adeno-associated virus. The first type encodes a portion of Factor VIII operably linked to an expression control element; and the second type encodes a different portion of Factor VIII operably linked to an expression control element. The first and second nucleotide sequences collectively encode a functional Factor VIII protein. Another form of the composition is a recombinant adeno-associated virus containing a nucleotide sequence encoding functional Factor VIII light or heavy chain operably linked to a tissue-specific promoter.

RELATED APPLICATIONS

[0001] This Application is a Continuation of U.S. application Ser. No.10/007,968, filed Nov. 16, 2001, now abandoned, which is a divisional ofU.S. application Ser. No. 09/740,211, filed Dec. 18, 2000, which is acontinuation of U.S. application Ser. No. 09/470,618, filed Dec. 22,1999, which is a continuation-in-part of U.S. application Ser. No.09/364,862, filed Jul. 30, 1999, which claims benefit under 35 U.S.C.§119(e) of U.S. Provisional Application Nos. 60/125,974 and 60/104,994,filed Mar. 24, 1999 and Oct. 20, 1998, respectively. All of these priorapplications are hereby incorporated by reference in their entireties.

FIELD OF THE INVENTION

[0002] The present invention relates to AAV vectors suitable forhemophilia gene therapy. More particularly, these AAV vectors aresuitable for delivering nucleic acids encoding Factor VIII into arecipient subject suffering from hemophilia A, such that the subject'sblood is able to clot.

DESCRIPTION OF THE RELATED ART

[0003] Hemophilia is a genetic disease characterized by a blood clottingdeficiency. In hemophilia A (classic hemophilia, Factor VIIIdeficiency), an X-chromosome-linked genetic defect disrupts the geneencoding Factor VIII, a plasma glycoprotein, which is a key component inthe blood clotting cascade. Human Factor VIII is synthesized as a singlechain polypeptide, with a predicted molecular weight of 265 kDa. TheFactor VIII gene codes for 2351 amino acids, and the protein has sixdomains, designated from the amino to the carboxy terminus asA1-A2-B-A3-C1-C2 (Wood et al., Nature 312:330 [1984]; Vehar et al.,Nature 312:337 [1984]; and Toole et al., Nature 312:342 [1984]). HumanFactor VIII is processed within the cell to yield a heterodimerprimarily comprised of a heavy chain of 200 kDa containing the A1, A2,and B domains and an 80 kDa light chain containing the A3, C1, and C2domains (Kaufman et al., J. Biol. Chem., 263:6352-6362 [1988]). Both thesingle chain polypeptide and the heterodimer circulate in the plasma asinactive precursors (Ganz et al., Eur. J. Biochem., 170:521-528 [1988]).Activation of Factor VIII in plasma is initiated by thrombin cleavagebetween the A2 and B domains, which releases the B domain and results ina heavy chain consisting of the A1 and A2 domains. The 980 amino acid Bdomain is deleted in the activated procoagulant form of the protein.Additionally, in the native protein, two polypeptide chains (“a” and“b”), flanking the B domain, are bound to a divalent calcium cation.Hemophilia may result from point mutations, deletions, or mutationsresulting in a stop codon (See, Antonarakis et al., Mol. Biol. Med.,4:81 [1987]).

[0004] The disease is relatively rare, afflicting approximately one in10,000 males. Hemophilia in females is extremely rare, although it mayoccur in female children of an affected father and carrier mother, aswell as in females with X-chromosomal abnormalities (e.g., Turnersyndrome, X mosaicism, etc.). The severity of each patient's disease isbroadly characterized into three groups—“mild,” “moderate,” and“severe,” depending on the severity of the patient's symptoms andcirculating Factor VIII levels. While normal levels of Factor VIII rangebetween 50 and 200 ng/mL plasma, mildly affected patients have 6-60% ofthis value, and moderately affected patients have 1-5% of this value.Severely affected hemophiliacs have less than 1% of normal Factor VIIIlevels.

[0005] While hemophiliacs clearly require clotting factor after surgeryor severe trauma, on a daily basis, spontaneous internal bleeding is agreater concern. Hemophiliacs experience spontaneous hemorrhages fromearly infancy, as well as frequent spontaneous hemarthroses and otherhemorrhages requiring clotting factor replacement. Without effectivetreatment, chronic hemophilic arthropathy occurs by young adulthood.Severely affected patients are prone to serious hemorrhages that maydissect through tissue planes, ultimately resulting in death due tocompromised vital organs.

[0006] Hematomas are commonly observed in moderately and severelyaffected hemophiliacs. In these patients, hematomas have a tendency toprogressively enlarge and dissect in all directions. Some of thesehematomas expand locally, resulting in local compression of adjacentorgans, blood vessels, and nerves. A rare, yet often fatal, complicationof abdominal hematomas is the perforation and drainage of the hematomainto the colon, resulting in infection and septicemia. Intracranialand/or extracranial hemorrhage also represent very dangerous bleedingsituations. While subcutaneous hematomas may dissect into muscle,pharyngeal and retropharyngeal hematomas (e.g., complicating bacterialor viral pharyngitis) may enlarge and obstruct the airway, sometimesresulting in a life-threatening situation that requires administrationof a sufficient dose of Factor VIII concentrate to normalize the FactorVIII level.

[0007] In addition to hematomas, hemarthroses are commonly observed inhemophiliacs, with bleeding into the joint accounting for approximately75% of hemophilic bleeding. Repeated hemorrhaging into the jointseventually results in extensive destruction of articular cartilage,synovial hyperplasia, and other reactive changes in adjacent tissues andbone. A major complication of repeated hemarthroses is joint deformity,which is often accompanied by muscle atrophy and soft tissuecontractures; osteoporosis and cystic areas in the subchondral bone mayalso develop, along with progressive loss of joint space.

[0008] Other symptoms are often observed in hemophiliacs, includinghematuria and mucous membrane bleeding. Hematuria is experienced byvirtually all severely affected hemophiliacs sometime during theirlifetimes, and mucous membrane bleeding is common in hemophiliacs. Bonecysts (pseudotumors) are rare, but dangerous complications of hemophilicbleeding. In many of these cases, immediate treatment is necessary.

[0009] In the early 1980s, many severely affected hemophiliacs weretreated with Factor VIII concentrate about three times weekly.Unfortunately, these concentrates transmitted viruses, such as hepatitisB and/or C, and human immunodeficiency virus (HIV). In the United Statesand Western Europe, at least 75% of Factor VIII concentrate recipientshave been reported to have anti-HIV antibodies (See, Schrier and Leung,supra). Some of these patients also developed HIV-associated immunethrombocytopenia, a very serious complication in hemophiliacs. In spiteof antiviral therapy (e.g., with zidovudine and pentamidineprophylaxis), which has tended to slow disease progression, full-blownAIDS (acquired immunodeficiency syndrome) occurs at an inexorable ratein hemophiliacs infected with HIV. Indeed, this has reversed theimprovement in the life expectancy of hemophiliacs, which peaked at 66years of age during the 1970s, and has dropped to 49 years (See, Schrierand Leung, supra). The development of virus-free preparations andrecombinant Factor VIII has helped control infectious viralcontamination.

[0010] However, for hemophiliacs, the availability of viral-freeconcentrates and recombinant Factor VIII, while significant, is but partof the solution. In order to prevent spontaneous internal bleedingepisodes, patients suffering from hemophilia A must consistently haveserum Factor VIII levels of about 1%, and preferably 5%. Currently, thecost of viral-free concentrates and recombinant Factor VIII make itprohibitively expensive to administer the clotting factorprophylactically or on a maintenance basis. Indeed, most hemophiliacs inthe U.S. do not receive recombinant Factor VIII therapy on a maintenancebasis, but only receive it prior to activities or events which mightcause bleeding (e.g., surgery), or as a treatment for spontaneousbleeding.

[0011] Moreover, even if cost effective preparations of recombinant orvirus-free Factor VIII were available, a steady state level of FactorVIII cannot be achieved by its daily administration. At best, patientsreceive widely varying levels of Factor VIII. Immediately following theadministration, the levels are super-physiological, while prior toadministration the levels are sub-physiological. Thus, there remains aneed for methods and compositions that are relatively economic, yeteffective in the treatment and prevention of bleeding in hemophiliacs,particularly spontaneous bleeds. Furthermore, there is a need in the artfor methods and compositions for long term delivery of clotting factors(e.g., Factor VIII) which more closely mimic the steady statephysiological levels observed in normal individuals.

SUMMARY OF THE INVENTION

[0012] The present invention provides improved viral vectors suitablefor gene therapy to treat hemophilia. In particular, the presentinvention provides AAV vectors and methods for treating hemophilia A bydelivering nucleic acids coding for the clotting protein Factor VIII.The present invention also provides pharmaceutical compositionscomprising such AAV vectors, as well as methods for making and using thevectors.

[0013] The present invention is particularly suited for use inhemophilia A gene therapy. Accordingly, in one embodiment of theinvention, at least one AAV vector containing a nucleic acid moleculeencoding Factor VIII is operably linked to control sequences that directexpression of Factor VIII in a suitable recipient cell. The AAV vectorsare then introduced into a recipient cell of the subject, underconditions that result in expression of Factor VIII. The subject,therefore, has a continuous supply of Factor VIII available to clotblood during bleeding episodes.

[0014] Using the methods of the present invention, long term expressionof therapeutic levels of Factor VIII have been achieved in vivo. In oneembodiment, animals were administered, via the portal vein, two AAVvectors: one carrying the DNA sequence coding for the heavy chain ofFactor VIII and the other carrying the DNA sequence coding for the lightchain of Factor VIII. Blood samples were collected periodically andassayed for Factor VIII activity. Reproducibly, animals expressedbetween 600 and 900 ng/ml of biologically active Factor VIII, levelsthat are well above the normal physiological levels of approximately 200ng/ml. Furthermore, these levels have been sustained for over 13 monthswithout a decrease in Factor VIII levels or activity. In a relatedembodiment, a B-domain deleted form of Factor VIII was cloned into asingle AAV vector and shown to express biologically active Factor VIII.

[0015] It is not intended, however, that the present invention belimited to specific embodiments. Many different forms of recombinantFactor VIII have been made and tested both in vitro and in vivo, using avariety of different control and regulatory sequences. Any DNA sequencecoding for biologically active Factor VIII can be expressed using theAAV vectors and methods taught in the present invention. Therefore, thepresent invention encompasses any AAV vector or vectors containingFactor VIII sequences that produce biologically active Factor VIIIprotein in vitro or in vivo.

[0016] For example, in some embodiments, the AAV vector contains thefirst 57 base pairs of the Factor VIII heavy chain which encodes the 10amino acid signal sequence, as well as the human growth hormone (hGH)polyadenylation sequence. In some alternative embodiments, the vectoralso contains the A1 and A2 domains, as well as 5 amino acids from theN-terminus of the B domain, and/or 85 amino acids of the C-terminus ofthe B domain, as well as the A3, C1, and C2 domains. In yet otherembodiments, the nucleic acids coding for Factor VIII heavy chain andlight chain were cloned into a single vector separated by 42 nucleicacids coding for 14 amino acids of the B domain.

[0017] The present invention also provides methods for administering theabove-described vectors. For example, it is intended that the presentinvention encompass methods suitable for delivery of the AAV vectors tothe livers of recipient patients or test animals. It is not intendedthat the present invention be limited to any particular route ofadministration. However, in preferred embodiments, the AAV vectors ofthe present invention are successfully administered via the portal orarterial vasculature.

[0018] These and other embodiments of the invention will readily occurto those of ordinary skill in the art in view of the disclosure herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019]FIG. 1 provides a schematic representation of the Factor VIIIprotein.

[0020]FIG. 2 provides a schematic representation of a B-domain deletedform of Factor VIII protein.

[0021]FIG. 3 provides a schematic representation of a B-domain deletedFactor VIII AAV construct (AAV-F8-1) from internal terminal repeat (ITRto ITR), including control sequences.

[0022]FIG. 4 provides a schematic representation of a B-domain deletedFactor VIII AAV construct (PVM4.1c-F8AB) from internal terminal repeat(ITR to ITR), including control sequences.

[0023]FIG. 5 provides the sequence of pAAV-F8-1 (ITR to ITR), with theplasmid backbone omitted.

[0024]FIG. 6 provides the sequence of pVm4.1cF8ΔB (ITR to ITR), with theplasmid backbone omitted.

[0025]FIG. 7 provides a map of rAAV-hFVIII-HC and rAAV-hFVIII-LCvectors.

[0026]FIG. 8 provides a graph demonstrating the expression of varioushuman FVIII constructs in mouse plasma.

[0027]FIG. 9 provides Southern blot analyses of liver DNA using probesspecific for (A) the light chain of hFVIII, and (B) the heavy chain ofhFVIII.

[0028]FIG. 10 provides Southern blot analyses of DNA from differenttissues using probes specific for (A) the light chain of hFVIII, and (B)the heavy chain of hFVIII.

[0029]FIG. 11 provides Northern blot analyses of liver RNA using probesspecific for (A) the light chain of hFVIII, and (B) the heavy chain ofhFVIII.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0030] The present invention relates to improved viral vectors usefulfor expressing gene products at high levels in human cells. Inparticular, the present invention provides AAV vectors suitable for genetherapy. These vectors are capable of delivering nucleic acid containingconstructs which result in the production of Factor VIII protein in ahost. The present invention also provides pharmaceutical compositionscomprising such AAV vectors, as well as methods for making and using theconstructs.

[0031] The AAV vectors and rAAV virions of the present invention can beproduced using standard methodology known to those of skill in the art.The methods generally involve the steps of: (1) introducing an AAVvector into a host cell; (2) introducing an AAV helper construct intothe host cell, where the helper construct includes AAV coding regionscapable of being expressed in the host cell to complement AAV helperfunctions missing from the AAV vector; (3) introducing one or morehelper viruses and/or accessory function vectors into the host cell,wherein the helper virus and/or accessory function vectors provideaccessory functions capable of supporting efficient recombinant AAV(“rAAV”) virion production in the host cell; and (4) culturing the hostcell to produce rAAV virions. The AAV vector, AAV helper construct andthe helper virus or accessory function vector(s) can be introduced intothe host cell either simultaneously or serially, using standardtransfection techniques.

[0032] Unless otherwise indicated, the practice of the present inventionemploys conventional methods of virology, microbiology, molecularbiology and recombinant DNA techniques within the skill of the art,including those described in such references as Sambrook et al. (eds.)Molecular Cloning: A Laboratory Manual; Glover (ed.) DNA Cloning: APractical Approach, Vols. I and II; Gait (ed.) OligonucleotideSynthesis; Hames and Higgins (eds.) Nucleic Acid Hybridization; Hamesand Higgins (eds.) Transcription and Translation; Tijessen (ed.) CRCHandbook of Parvoviruses, Vols. I and II; and Fields and Knipe (eds.)Fundamental Virology, 2nd Edition, Vols. I and II.

Definitions

[0033] In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

[0034] As used herein, the terms “gene transfer” and “gene delivery”refer to methods or systems for reliably inserting a particularnucleotide sequence (e.g., DNA) into targeted cells. In particularlypreferred embodiments, the nucleotide sequence comprises at least aportion of Factor VIII.

[0035] As used herein, the terms “vector,” and “gene transfer vector”refer to any genetic element, such as a plasmid, phage, transposon,cosmid, chromosome, virus, virion, etc., which is capable of replicationwhen associated with the proper control sequences and/or which cantransfer nucleic acid sequences between cells. Thus, the term includescloning and expression vectors, as well as viral vectors.

[0036] Gene transfer vectors may include transcription sequences such aspolyadenylation sites, selectable markers or reporter genes, enhancersequences, and other control sequences which allow for the induction oftranscription. Such control sequences are described more fully below.

[0037] The term “expression vector” as used herein refers to arecombinant DNA molecule containing a desired coding sequence andappropriate nucleic acid sequences necessary for the expression of theoperably linked coding sequence in a particular host organism. Nucleicacid sequences necessary for expression in prokaryotes usually include apromoter, an operator (optional), and a ribosome binding site, as wellas other sequences. Eukaryotic cells are generally known to utilizepromoters (constitutive, inducible or tissue specific), enhancers, andtermination and polyadenylation signals, although some elements may bedeleted and other elements added without sacrificing the necessaryexpression.

[0038] As used herein, the terms “host” and “expression host” refer toorganisms and/or cells which harbor an exogenous DNA sequence (e.g., viatransfection), an expression vector or vehicle, as well as organismsand/or cells that are suitable for use in expressing a recombinant geneor protein. It is not intended that the present invention be limited toany particular type of cell or organism. Indeed, it is contemplated thatany suitable organism and/or cell will find use in the present inventionas a host.

[0039] As used herein, the terms “viral replicons” and “viral origins ofreplication” refer to viral DNA sequences that allow for theextrachromosomal replication of a vector in a host cell expressing theappropriate replication factors. In some embodiments, vectors whichcontain either the SV40 or polyoma virus origin of replication replicateto high copy number, while vectors which contain the replicons frombovine papillomavirus or Epstein-Barr virus replicate extrachromosomallyat low copy number may be utilized in other embodiments.

[0040] As used herein, the term “AAV vector” refers to a vector havingfunctional or partly functional ITR sequences. The ITR sequences may bederived from an adeno-associated virus serotype, including withoutlimitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-X7, etc. The ITRs,however, need not be the wild-type nucleotide sequences, and may bealtered (e.g., by the insertion, deletion or substitution ofnucleotides), so long as the sequences retain function provide forfunctional rescue, replication and packaging. AAV vectors can have oneor more of the AAV wild-type genes deleted in whole or part, preferablythe rep and/or cap genes but retain functional flanking ITR sequences.Functional ITR sequences are necessary for the rescue, replication andpackaging of the AAV virion. Thus, an “AAV vector” is defined herein toinclude at least those sequences required in cis for replication andpackaging (e.g., functional ITRs) of the virus.

[0041] As used herein, the term “ITR” refers to inverted terminalrepeats. The terms “adeno-associated virus inverted terminal repeats” or“AAV ITRs” refer to the art-recognized palindromic regions found at eachend of the AAV genome which function together in cis as origins of DNAreplication and as packaging signals for the virus. For use in someembodiments of the present invention, flanking AAV ITRs are positioned5′ and 3′ of one or more selected heterologous nucleotide sequences.Optionally, the ITRs together with the rep coding region or the Repexpression product provide for the integration of the selected sequencesinto the genome of a target cell.

[0042] As used herein, the term “AAV rep coding region” refers to theart-recognized region of the AAV genome which encodes the replicationproteins Rep 78, Rep 68, Rep 52 and Rep 40. These Rep expressionproducts have been shown to possess many functions, includingrecognition, binding and nicking of the AAV origin of DNA replication,DNA helicase activity and modulation of transcription from AAV (or otherheterologous) promoters. The Rep expression products are collectivelyrequired for replicating the AAV genome. Muzyczka (Muzyczka, Curr. Top.Microbiol. Immunol., 158:97-129 [1992]) and Kotin (Kotin, Hum. GeneTher., 5:793-801 [1994]) provide additional descriptions of the AAV repcoding region, as well as the cap coding region described below.Suitable homologues of the AAV rep coding region include the humanherpesvirus 6 (HHV-6) rep gene which is also known to mediate AAV-2 DNAreplication (Thomson et al., Virol., 204:304-311 [1994]).

[0043] As used herein, the term “AAV cap coding region” refers to theart-recognized region of the AAV genome which encodes the capsidproteins VP1, VP2, and VP3, or functional homologues thereof. These capexpression products supply the packaging functions which arecollectively required for packaging the viral genome.

[0044] As used herein, the term “AAV helper function” refers to AAVcoding regions capable of being expressed in the host cell to complementAAV viral functions missing from the AAV vector. Typically, the AAVhelper functions include the AAV rep coding region and the AAV capcoding region. An “AAV helper construct” is a vector containing AAVcoding regions required to complement AAV viral functions missing fromthe AAV vector (e.g., the AAV rep coding region and the AAV cap codingregion).

[0045] As used herein, the terms “accessory functions” and “accessoryfactors” refer to functions and factors that are required by AAV forreplication, but are not provided by the AAV virion (or rAAV virion)itself. Thus, these accessory functions and factors must be provided bythe host cell, a virus (e.g., adenovirus or herpes simplex virus), oranother expression vector that is co-expressed in the same cell.Generally, the E1, E2A, E4 and VA coding regions of adenovirus are usedto supply the necessary accessory function required for AAV replicationand packaging (Matsushita et al., Gene Therapy 5:938 [1998]).

[0046] As used herein, the term “wild type” (“wt”) refers to a gene orgene product which has the characteristics of that gene or gene productwhen isolated from a naturally occurring source. A wild-type gene isthat which is most frequently observed in a population and is thusarbitrarily designed the “normal” or “wild-type” form of the gene. Incontrast, the term “modified” or “mutant” refers to a gene or geneproduct which displays modifications in sequence and or functionalproperties (i.e., altered characteristics) when compared to thewild-type gene or gene product. It is noted that naturally-occurringmutants can be isolated; these are identified by the fact that they havealtered characteristics when compared to the wild-type gene or geneproduct.

[0047] As used herein, the term “AAV virion” refers to a complete virusparticle, such as a “wild-type” (wt) AAV virus particle (comprising alinear, single-stranded AAV nucleic acid genome associated with an AAVcapsid protein coat). In this regard, single-stranded AAV nucleic acidmolecules of either complementary sense (e.g., “sense” or “antisense”strands), can be packaged into any one AAV virion and both strands areequally infectious.

[0048] As used herein, the terms “recombinant AAV virion,” and “rAAVvirion” refer to an infectious viral particle containing a heterologousDNA molecule of interest (e.g., Factor VIII sequence) which is flankedon both sides by AAV ITRs. In some embodiments of the present invention,an rAAV virion is produced in a suitable host cell which contains an AAVvector, AAV helper functions and accessory functions introduced therein.In this manner, the host cell is rendered capable of encoding AAVpolypeptides that are required for packaging the AAV vector containing arecombinant nucleotide sequence of interest, such as at least a portionof Factor VIII or portions of Factor VIII domains, into recombinantvirion particles for subsequent gene delivery.

[0049] As used herein, the term “transfection” refers to the uptake offoreign DNA by a cell, and a cell has been “transfected” when exogenousDNA has been introduced inside the cell membrane. A number oftransfection techniques are generally known in the art (See e.g., Grahamet al., Virol., 52:456 [1973]; Sambrook et al., Molecular Cloning, aLaboratory Manual, Cold Spring Harbor Laboratories, New York [1989];Davis et al., Basic Methods in Molecular Biology, Elsevier, [1986]; andChu et al., Gene 13:197 [1981]. Such techniques can be used to introduceone or more exogenous DNA moieties, such as a gene transfer vector andother nucleic acid molecules, into suitable recipient cells.

[0050] As used herein, the terms “stable transfection” and “stablytransfected” refers to the introduction and integration of foreign DNAinto the genome of the transfected cell. The term “stable transfectant”refers to a cell which has stably integrated foreign DNA into thegenomic DNA.

[0051] As used herein, the term “transient transfection” or “transientlytransfected” refers to the introduction of foreign DNA into a cell wherethe foreign DNA fails to integrate into the genome of the transfectedcell. The foreign DNA persists in the nucleus of the transfected cellfor several days. During this time the foreign DNA is subject to theregulatory controls that govern the expression of endogenous genes inthe chromosomes. The term “transient transfectant” refers to cells whichhave taken up foreign DNA but have failed to integrate this DNA.

[0052] As used herein, the term “transduction” denotes the delivery of aDNA molecule to a recipient cell either in vivo or in vitro, via areplication-defective viral vector, such as via a recombinant AAVvirion.

[0053] As used herein, the term “recipient cell” refers to a cell whichhas been transfected or transduced, or is capable of being transfectedor transduced, by a nucleic acid construct or vector bearing a selectednucleotide sequence of interest (i.e., Factor VIII). The term includesthe progeny of the parent cell, whether or not the progeny are identicalin morphology or in genetic make-up to the original parent, so long asthe selected nucleotide sequence is present.

[0054] The term “heterologous” as it relates to nucleic acid sequencessuch as coding sequences and control sequences, denotes sequences thatare not normally joined together, and/or are not normally associatedwith a particular cell. Thus, a “heterologous” region of a nucleic acidconstruct or a vector is a segment of nucleic acid within or attached toanother nucleic acid molecule that is not found in association with theother molecule in nature. For example, a heterologous region of anucleic acid construct could include a coding sequence flanked bysequences not found in association with the coding sequence in nature.Another example of a heterologous coding sequence is a construct wherethe coding sequence itself is not found in nature (e.g., syntheticsequences having codons different from the native gene). Similarly, acell transfected with a construct which is not normally present in thecell would be considered heterologous for purposes of this invention.Allelic variation or naturally occurring mutational events do not giverise to heterologous DNA, as used herein.

[0055] As used herein, “coding sequence” or a sequence which “encodes” aparticular antigen, is a nucleic acid sequence which is transcribed (inthe case of DNA) and translated (in the case of mRNA) into a polypeptidein vitro or in vivo, when placed under the control of appropriateregulatory sequences. The boundaries of the coding sequence aredetermined by a start codon at the 5′ (amino) terminus and a translationstop codon at the 3′ (carboxy) terminus. A coding sequence can include,but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomicDNA sequences from prokaryotic or eukaryotic DNA, and even synthetic DNAsequences. A transcription termination sequence will usually be located3′ to the coding sequence.

[0056] As used herein, the term “nucleic acid” sequence refers to a DNAor RNA sequence. The term captures sequences that include any of theknown base analogues of DNA and RNA such as, but not limited to4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine,pseudoisocytosine, 5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil,5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethylaminomethyluracil, dihydrouracil, inosine,N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and2,6-diaminopurine.

[0057] As used herein, the term “recombinant DNA molecule” as usedherein refers to a DNA molecule which is comprised of segments of DNAjoined together by means of molecular biological techniques.

[0058] As used herein, the term “regulatory element” refers to a geneticelement which controls some aspect of the expression of nucleic acidsequences. For example, a promoter is a regulatory element whichfacilitates the initiation of transcription of an operably linked codingregion. Other regulatory elements are splicing signals, polyadenylationsignals, termination signals, etc. (defined infra).

[0059] The term DNA “control sequences” refers collectively toregulatory elements such as promoter sequences, polyadenylation signals,transcription termination sequences, upstream regulatory domains,origins of replication, internal ribosome entry sites (“IRES”),enhancers, and the like, which collectively provide for the replication,transcription and translation of a coding sequence in a recipient cell.Not all of these control sequences need always be present so long as theselected coding sequence is capable of being replicated, transcribed andtranslated in an appropriate recipient cell.

[0060] Transcriptional control signals in eukaryotes generally comprise“promoter” and “enhancer” elements. Promoters and enhancers consist ofshort arrays of DNA sequences that interact specifically with cellularproteins involved in transcription (Maniatis et al., Science 236:1237[1987]). Promoter and enhancer elements have been isolated from avariety of eukaryotic sources including genes in yeast, insect andmammalian cells and viruses (analogous control sequences, i.e.,promoters, are also found in prokaryotes). The selection of a particularpromoter and enhancer depends on what cell type is to be used to expressthe protein of interest (i.e., Factor VIII). Some eukaryotic promotersand enhancers have a broad host range while others are functional in alimited subset of cell types (See e.g., Voss et al., Trends Biochem.Sci., 11:287 [1986]; and Maniatis et al., supra, for reviews). Forexample, the SV40 early gene enhancer is very active in a wide varietyof cell types from many mammalian species and has been widely used forthe expression of proteins in mammalian cells (Dijkema et al., EMBO J.4:761 [1985]). Two other examples of promoter and enhancer elementsactive in a broad range of mammalian cell types are those from the humanelongation factor 1a gene (Uetsuki et al., J. Biol. Chem., 264:5791[1989]; Kim et al., Gene 91:217 [1990]; and Mizushima and Nagata, Nucl.Acids. Res., 18:5322 [1990]) and the long terminal repeats of the Roussarcoma virus (Gorman et al., Proc. Natl. Acad. Sci. USA 79:6777 [1982])and the human cytomegalovirus (Boshart et al., Cell 41:521 [1985]).Promoters and enhances can be found naturally alone or together. Forexample, the long terminal repeats of retroviruses contain both promoterand enhancer functions Moreover, generally promoters and enhances actindependently of the gene being transcribed or translated. Thus, theenhancer and promoter may be “endogenous” or “exogenous” or“heterologous.” An “endogenous” enhancer/promoter is one which isnaturally linked with a given gene in the genome. An “exogenous” or“heterologous” enhancer and promoter is one which is placed injuxtaposition to a gene by means of genetic manipulation (i.e.,molecular biological techniques) such that transcription of that gene isdirected by the linked enhancer/promoter.

[0061] As used herein, the term “tissue specific” refers to regulatoryelements or control sequences, such as a promoter, enhancers, etc.,wherein the expression of the nucleic acid sequence is substantiallygreater in a specific cell type(s) or tissue(s). In particularlypreferred embodiments, the albumin promoter and the transthyretinpromoter display increased expression of FVIII in hepatocytes, ascompared to other cell types. It is not intended, however, that thepresent invention be limited to the albumin or transthyretin promotersor to hepatic-specific expression, as other tissue specific regulatoryelements, or regulatory elements that display altered gene expressionpatterns, are contemplated.

[0062] The presence of “splicing signals” on an expression vector oftenresults in higher levels of expression of the recombinant transcript.Splicing signals mediate the removal of introns from the primary RNAtranscript and consist of a splice donor and acceptor site (Sambrook etal., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory Press, New York [1989], pp. 16.7-16.8). A commonly usedsplice donor and acceptor site is the splice junction from the 16S RNAof SV40.

[0063] Efficient expression of recombinant DNA sequences in eukaryoticcells requires expression of signals directing the efficient terminationand polyadenylation of the resulting transcript. Transcriptiontermination signals are generally found downstream of thepolyadenylation signal and are a few hundred nucleotides in length. Theterm “poly A site” or “poly A sequence” as used herein denotes a DNAsequence which directs both the termination and polyadenylation of thenascent RNA transcript. Efficient polyadenylation of the recombinanttranscript is desirable as transcripts lacking a poly A tail areunstable and are rapidly degraded. The poly A signal utilized in anexpression vector may be “heterologous” or “endogenous.” An endogenouspoly A signal is one that is found naturally at the 3′ end of the codingregion of a given gene in the genome. A heterologous poly A signal isone which is one which is isolated from one gene and placed 3′ ofanother gene. A commonly used heterologous poly A signal is the SV40poly A signal. The SV40 poly A signal is contained on a 237 bpBamHI/BclI restriction fragment and directs both termination andpolyadenylation (Sambrook et al., supra, at 16.6-16.7).

[0064] “Operably linked” refers to an arrangement of elements whereinthe components so described are configured so as to perform their usualfunction. Thus, control sequences operably linked to a coding sequenceare capable of effecting the expression of the coding sequence. Thecontrol sequences need not be contiguous with the coding sequence, solong as they function to direct the expression thereof. Thus, forexample, intervening untranslated yet transcribed sequences can bepresent between a promoter sequence and the coding sequence and thepromoter sequence can still be considered “operably linked” to thecoding sequence.

[0065] term “isolated” when used in relation to a nucleic acid, as in“an isolated oligonucleotide” or “isolated polynucleotide” refers to anucleic acid sequence that is identified and separated from at least onecontaminant nucleic acid with which it is ordinarily associated in itsnatural source. Isolated nucleic acid is such present in a form orsetting that is different from that in which it is found in nature. Incontrast, non-isolated nucleic acids are nucleic acids such as DNA andRNA found in the state they exist in nature. For example, a given DNAsequence (e.g., a gene) is found on the host cell chromosome inproximity to neighboring genes; RNA sequences, such as a specific mRNAsequence encoding a specific protein, are found in the cell as a mixturewith numerous other mRNAs which encode a multitude of proteins. Theisolated nucleic acid, oligonucleotide, or polynucleotide may be presentin single-stranded or double-stranded form. When an isolated nucleicacid, oligonucleotide or polynucleotide is to be utilized to express aprotein, the oligonucleotide or polynucleotide will contain at a minimumthe sense or coding strand (i.e., the oligonucleotide or polynucleotidemay single-stranded), but may contain both the sense and anti-sensestrands (i.e., the oligonucleotide or polynucleotide may bedouble-stranded).

[0066] As used herein, the term “purified” or “to purify” refers to theremoval of contaminants from a sample. For example, antibodies may bepurified by removal of contaminating non-immunoglobulin proteins; theymay also purified by the removal of immunoglobulin that does not bindthe antigen of interest (e.g., at least a portion of Factor VIII). Theremoval of non-immunoglobulin proteins and/or the removal ofimmunoglobulins that do not bind the antigen of interest (e.g., at leasta portion of Factor VIII) results in an increase in the percent ofdesired antigen-reactive immunoglobulins in the sample. In anotherexample, recombinant polypeptides of Factor VIII are expressed inbacterial host cells and the polypeptides are purified by the removal ofhost cell proteins; the percent of recombinant polypeptides is therebyincreased in the sample.

[0067] As used herein, the term “chimeric protein” refers to two or morecoding sequences obtained from different genes, that have been clonedtogether and that, after translation, act as a single polypeptidesequence. Chimeric proteins are also referred to as “hybrid proteins.”As used herein, the term “chimeric protein” refers to coding sequencesthat are obtained from different species of organisms, as well as codingsequences that are obtained from the same species of organisms.

[0068] A “composition comprising a given polynucleotide sequence” asused herein refers broadly to any composition containing the givenpolynucleotide sequence. The composition may comprise an aqueoussolution.

[0069] As used herein, the term “at risk” is used in references toindividuals who are at risk for experiencing hemorrhagic episodes. Inparticularly preferred embodiments, the individuals are hemophiliacswith mild, moderate, or severe hemophilia.

[0070] As used herein, the term “subject” refers to any animal (i.e.,vertebrates and invertebrates), while the term “vertebrate subject”refers to any member of the subphylum Chordata. It is intended that theterm encompass any member of this subphylum, including, but not limitedto humans and other primates, rodents (e.g., mice, rats, and guineapigs), lagamorphs (e.g., rabbits), bovines (e.g., cattle), ovines (e.g.,sheep), caprines (e.g., goats), porcines (e.g., swine), equines (e.g.,horses), canines (e.g., dogs), felines (e.g., cats), domestic fowl(e.g., chickens, turkeys, ducks, geese, other gallinaceous birds, etc.),as well as feral or wild animals, including, but not limited to, suchanimals as ungulates (e.g., deer), bear, fish, lagamorphs, rodents,birds, etc. It is not intended that the term be limited to a particularage or sex. Thus, adult and newborn subjects, as well as fetuses,whether male or female, are encompassed by the term.

[0071] As defined herein, a “therapeutically effective amount” or“therapeutic effective dose” is an amount or dose of AAV vector orvirions capable of producing sufficient amounts of Factor VIII todecrease the time it takes for a subject's blood to clot. Generally,severe hemophiliacs having less than 1% of normal levels of FVIII have awhole blood clotting time of greater than 60 minutes as compared toapproximately 10 minutes for non-hemophiliacs.

[0072] The present invention relates to AAV vectors suitable forhemophilia A gene therapy. More particularly, these AAV vectors aresuitable for delivering nucleic acids encoding Factor VIII into arecipient host suspected of suffering from a blood clotting disorder.Using the nucleic acid as a template, the host produces Factor VIII,such that the subject's blood is able to clot. The present inventionalso provides pharmaceutical compositions comprising such AAV vectors,as well as methods for making and using the constructs.

[0073] I. AAV Vectors

[0074] Adeno-associated virus (AAV) is a non-pathogenic,replication-defective, helper-dependent parvovirus (or “dependovirus” or“adeno-satellite virus”). There are at least six recognized serotypes,designated as AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-X7, etc. Cultureand serologic evidence indicates that human infection occurs with AAV-2and AAV-3. Although 85% of the human population is seropositive forAAV-2, the virus has never been associated with disease in humans.Recombinant AAV (rAAV) virions are of interest as vectors for genetherapy because of their broad host range, excellent safety profile, andduration of transgene expression in infected hosts. One remarkablefeature of recombinant AAV (rAAV) virions is the prolonged expressionachieved after in vivo administration.

[0075] AAV vectors of the present invention may be constructed usingknown techniques to provide, as operatively linked components in thedirection of transcription, (a) control sequences including atranscriptional initiation and termination regions, and (b) a nucleotidesequence encoding at least a portion of Factor VIII. The controlsequences are selected to be functional in a targeted recipient cell.The resulting construct which contains the operatively linked componentsis bounded (5′ and 3′) with functional AAV ITR sequences.

[0076] The nucleotide sequences of AAV ITR regions are known (See e.g.,Kotin, Hum. Gene Ther., 5:793-801 [1994]; Berns, “Parvoviridae and TheirReplication” in Fields and Knipe (eds), Fundamental Virology, 2ndEdition, for the AAV-2 sequence). AAV ITRs used in the vectors of theinvention need not have a wild-type nucleotide sequence, and may bealtered (e.g., by the insertion, deletion or substitution ofnucleotides). Additionally, AAV ITRs may be derived from any of severalAAV serotypes, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4,AAV-5, AAVX7, etc. Furthermore, 5′ and 3′ ITRs which flank a selectednucleotide sequence in an AAV vector need not necessarily be identicalor derived from the same AAV serotype or isolate, so long as theyfunction as intended.

[0077] A. Control Sequences

[0078] In some embodiments of the present invention, heterologouscontrol sequences are employed with the vectors. Useful heterologouscontrol sequences generally include those derived from sequencesencoding mammalian or viral genes. Examples include, but are not limitedto, the SV40 early promoter, mouse mammary tumor virus LTR promoter,adenovirus major late promoter (Ad MLP), a herpes simplex virus (HSV)promoter, a cytomegalovirus (CMV) promoter such as the CMV immediateearly promoter region (CMVIE), a rous sarcoma virus (RSV) promoter,synthetic promoters, hybrid promoters, and the like. In addition,sequences derived from nonviral genes, such as the murinemetallothionein gene, also find use herein. Such promoter sequences arecommercially available (e.g., from Stratagene).

[0079] It is contemplated that in some embodiments, tissue-specificexpression may be desirable (e.g., expression of biologically activeFactor VIII by hepatocytes). It is not intended that the presentinvention be limited to expression of biologically active Factor VIII byany particular cells or cell types. However, as hepatocytes (i.e., livercells) are the cells that normally synthesized Factor VIII (See,Kaufman, Ann. Rev. Med., 43:325 [1992]), it is contemplated that in someparticularly preferred embodiments, the compositions of the presentinvention be administered to the liver.

[0080] In preferred embodiments, expression is achieved by coupling thecoding sequence for Factor VIII with heterologous control sequencesderived from genes that are specifically transcribed by a selectedtissue type. A number of tissue-specific promoters have been describedabove which enable directed expression in selected tissue types.However, control sequences used in the present AAV vectors can alsocomprise control sequences normally associated with the selected nucleicacid sequences.

[0081] B. Construction of AAV Factor VIII Vectors

[0082] AAV vectors that contain a control sequence and a nucleotidesequence of interest (i.e., at least a portion of the sequence encodingFactor VIII), bounded by AAV ITRs (i.e., AAV vectors), can beconstructed by directly inserting selected sequences into an AAV genomewith the major AAV open reading frames (“ORFs”) excised. Other portionsof the AAV genome can also be deleted, so long as a sufficient portionof the ITRs remain to allow for replication and packaging functions.These constructs can be designed using techniques well known in the art(See e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941, all of which areherein incorporated by reference); International Publication Nos. WO92/01070 and WO 93/03769; Lebkowski et al., Mol. Cell. Biol.,8:3988-3996 [1988]; Vincent et al., Vaccines 90 [Cold Spring HarborLaboratory Press, 1990]; Carter, Curr. Opin. Biotechnol., 3:533-539[1992]; Muzyczka, Curr. Top. Microbiol. Immunol., 158:97-129 [1992];Kotin, Hum. Gene Ther., 5:793-801 [1994]; Shelling and Smith, GeneTher., 1:165-169 [1994]; and Zhou et al., J. Exp. Med., 179:1867-1875[1994]).

[0083] Alternatively, AAV ITRs can be excised from the viral genome orfrom an AAV vector containing the same and fused 5′ and 3′ of a selectednucleic acid construct that is present in another vector using standardligation techniques, such as those described in Sambrook et al., supra.For example, ligations can be accomplished in 20 mM Tris-Cl pH 7.5, 10mM MgCl₂, 10 mM DTT, 33 μg/ml BSA, 10 mM-50 mM NaCl, and either 40 μMATP, 0.01-0.02 (Weiss) units T4 DNA ligase at 0° C. (for “sticky end”ligation) or 1 mM ATP, 0.3-0.6 (Weiss) units T4 DNA ligase at 14° C.(for “blunt end” ligation). Intermolecular “sticky end” ligations areusually performed at 30-100 μg/ml total DNA concentrations (5-100 nMtotal end concentration). AAV vectors which contain ITRs have beendescribed in (e.g., U.S. Pat. No. 5,139,941, herein incorporated byreference). In particular, several AAV vectors are described thereinwhich are available from the American Type Culture Collection (“ATCC”)under Accession Numbers 53222, 53223, 53224, 53225 and 53226.

[0084] Additionally, chimeric genes can be produced synthetically toinclude AAV ITR sequences arranged 5′ and 3′ of a selected nucleic acidsequence. The complete chimeric sequence is assembled from overlappingoligonucleotides prepared by standard methods (See e.g., Edge, Nature292:756 [1981]; Nambair et al., Science 223:1299 [1984]; and Jay et al.,J. Biol. Chem., 259:6311 [1984]).

[0085] Moreover, it is not intended that the present invention belimited to any specific Factor VIII sequence. Many natural andrecombinant forms of Factor VIII have been isolated and assayed both invitro and in vivo, using a variety of different regulatory elements andcontrol sequences. Therefore, any known, or later discovered, DNAsequence coding for biologically active Factor VIII can be expressed,alone or in combination with at least one additional vector, using theAAV vectors and methods taught in the present invention. Examples ofnaturally occurring and recombinant forms of Factor VIII can be found inthe patent and scientific literature including, U.S. Pat. No. 5,563,045,U.S. Pat. No. 5,451,521, U.S. Pat. No. 5,422,260, U.S. Pat. No.5,004,803, U.S. Pat. No. 4,757,006, U.S. Pat. No. 5,661,008, U.S. Pat.No. 5,789,203, U.S. Pat. No. 5,681,746, U.S. 5,595,886, U.S. Pat. No.5,045,455, U.S. Pat. No. 5,668,108, U.S. Pat. No. 5,633,150, U.S. Pat.No. 5,693,499, U.S. Pat. No. 5,587,310, U.S. Pat. No. 5,171,844, U.S.Pat. No. 5,149,637, U.S. Pat. No. 5,112,950, U.S. Pat. No. 4,886,876, WO94/11503, WO 87/07144, WO 92/16557, WO 91/09122, WO 97/03195, WO96/21035, WO 91/07490, EP 0 672 138, EP 0 270 618, EP 0 182 448, EP 0162 067, EP 0 786 474, EP 0 533 862, EP 0 506 757, EP 0 874 057, EP 0795 021, EP 0 670 332, EP 0 500 734, EP 0 232 112, EP 0 160 457, Sanberget al., XXth Int. Congress of the World Fed. Of Hemophilia (1992), andLind et al., Eur. J. Biochem., 232:19 (1995).

[0086] Nucleic acid sequences coding for the above-described Factor VIIIcan be obtained using recombinant methods, such as by screening cDNA andgenomic libraries from cells expressing Factor VIII or by deriving thesequence from a vector known to include the same. Furthermore, thedesired sequence can be isolated directly from cells and tissuescontaining the same, using standard techniques, such as phenolextraction and PCR of cDNA or genomic DNA (See e.g., Sambrook et al.,supra, for a description of techniques used to obtain and isolate DNA).Nucleotide sequences encoding an antigen of interest (i.e., Factor VIIIsequence) can also be produced synthetically, rather than cloned. Thecomplete sequence can be assembled from overlapping oligonucleotidesprepared by standard methods and assembled into a complete codingsequence (See e.g., Edge, Nature 292:756 [1981]; Nambair et al., Science223:1299 [1984]; and Jay et al., J. Biol. Chem., 259:6311 [1984]).

[0087] Although it is not intended that the present invention be limitedto any particular methods for assessing the production of biologicallyactive Factor VIII, such methods as immunoassays (e.g., ELISA) andbiological activity assays are contemplated (e.g., coagulation activityassays).

[0088] Furthermore, while in particularly preferred embodiments, humanFactor VIII is encompassed by the present invention, it is not intendedthat the present invention be limited to human Factor VIII. Indeed, itis intended that the present invention encompass Factor VIII fromanimals other than humans, including but not limited to companionanimals (e.g., canines, felines, and equines), livestock (e.g., bovines,caprines, and ovines), laboratory animals (e.g., rodents such asmurines, as well as lagamorphs), and “exotic” animals (e.g., marinemammals, large cats, etc.).

[0089] II. Virion Production

[0090] Producing AAV Factor VIII vectors and rAAV Factor VIII virions ofthe present invention generally involve the steps of: (1) introducing anAAV vector containing the Factor VIII gene into a host cell; (2)introducing an AAV helper construct into the host cell, where the helperconstruct includes AAV coding regions capable of being expressed in thehost cell to complement AAV helper functions missing from the AAVvector; (3) introducing one or more helper viruses and/or accessoryfunction vectors into the host cell, wherein the helper virus and/oraccessory function vectors provide accessory functions capable ofsupporting efficient recombinant AAV (“rAAV”) virion production in thehost cell; and (4) culturing the host cell to produce rAAV virions.

[0091] The above-described vectors and constructs can be introduced intoa cell using standard methodology known to those of skill in the art(e.g., transfection). A number of transfection techniques are generallyknown in the art (See e.g., Graham et al., Virol., 52:456 [1973],Sambrook et al. supra, Davis et al., supra, and Chu et al., Gene 13:197

[0092] Particularly suitable transfection methods include calciumphosphate co-precipitation (Graham et al., Virol., 52:456-467 [1973]),direct micro-injection into cultured cells (Capecchi, Cell 22:479-488[1980]), electroporation (Shigekawa et al., BioTechn., 6:742-751[1988]), liposome-mediated gene transfer (Mannino et al., BioTechn.,6:682-690 [1988]) lipid-mediated transduction (Felgner et al., Proc.Natl. Acad. Sci. USA 84:7413-7417 [1987]), and nucleic acid deliveryusing high-velocity microprojectiles (Klein et al., Nature 327:70-73[1987]).

[0093] For the purposes of the invention, suitable host cells forproducing rAAV virions include microorganisms, yeast cells, insectcells, and mammalian cells, that can be, or have been, used asrecipients of a heterologous DNA molecule. The term includes the progenyof the original cell which has been transfected. Thus, as indicatedabove, a “host cell” as used herein generally refers to a cell which hasbeen transfected with an exogenous DNA sequence. Cells from the stablehuman cell line, 293 (ATCC Accession No. CRL1573) are preferred in thepractice of the present invention. Particularly, the human cell line 293is a human embryonic kidney cell line that has been transformed withadenovirus type-5 DNA fragments (Graham et al., J. Gen. Virol., 36:59[1977]), and expresses the adenoviral E1a and E1b genes (Aiello et al.,Virol., 94:460 [1979]). The 293 cell line is readily transfected, andprovides a particularly convenient platform in which to produce rAAVvirions.

[0094] Host cells containing the above-described AAV vectors must berendered capable of providing AAV helper functions in order to replicateand encapsidate the nucleotide sequences flanked by the AAV ITRs toproduce rAAV virons. AAV helper functions are generally AAV-derivedcoding sequences which can be expressed to provide AAV gene productsthat, in turn, function in trans for productive AAV replication. AAVhelper functions are used herein to complement necessary AAV functionsthat are missing from the AAV vectors. Thus, AAV helper functionsinclude one, or both of the major AAV ORFs, namely the rep and capcoding regions, or functional homologues thereof.

[0095] AAV helper functions are introduced into the host cell bytransfecting the host cell with an AAV helper construct either prior to,or concurrently with, the transfection of the AAV vector. AAV helperconstructs are thus used to provide at least transient expression of AAVrep and/or cap genes to complement missing AAV functions that arenecessary for productive AAV infection. AAV helper constructs lack AAVITRs and can neither replicate nor package themselves.

[0096] In preferred embodiments, these constructs are in the form of avector, including, but not limited to, plasmids, phages, transposons,cosmids, viruses, or virions. A number of AAV helper constructs havebeen described, such as the commonly used plasmids pAAV/Ad and pIM29+45which encode both Rep and Cap expression products (See e.g., Samulski etal., J. Virol,. 63:3822-3828 [1989]; and McCarty et al., J. Virol.,65:2936-2945

[0097] A number of other vectors have been described which encode Repand/or Cap expression products (See e.g., U.S. Pat. No. 5,139,941,herein incorporated by reference).

[0098] Both AAV vectors and AAV helper constructs can be constructed tocontain one or more optional selectable markers. Suitable markersinclude genes which confer antibiotic resistance or sensitivity to,impart color to, or change the antigenic characteristics of those cellswhich have been transfected with a nucleic acid construct containing theselectable marker when the cells are grown in an appropriate selectivemedium. Several selectable marker genes that are useful in the practiceof the invention include the gene encoding aminoglycosidephosphotranferase (APH) that allows selection in mammalian cells byconferring resistance to G418 (Sigma). Other suitable markers are knownto those of skill in the art.

[0099] The host cell (or packaging cell) must also be rendered capableof providing non-AAV derived functions, or “accessory functions,” inorder to produce rAAV virions. Accessory functions are non-AAV derivedviral and/or cellular functions upon which AAV is dependent for itsreplication. Thus, accessory functions include at least those non-AAVproteins and RNAs that are required in AAV replication, including thoseinvolved in activation of AAV gene transcription, stage specific AAVmRNA splicing, AAV DNA replication, synthesis of rep and cap expressionproducts and AAV capsid assembly. Viral-based accessory functions can bederived from any of the known helper viruses.

[0100] Particularly, accessory functions can be introduced into and thenexpressed in host cells using methods known to those of skill in theart. Commonly, accessory functions are provided by infection of the hostcells with an unrelated helper virus. A number of suitable helperviruses are known, including adenoviruses; herpesviruses such as herpessimplex virus types 1 and 2; and vaccinia viruses. Nonviral accessoryfunctions will also find use herein, such as those provided by cellsynchronization using any of various known agents (See e.g., Buller etal., J. Virol., 40:241-247 [1981]; McPherson et al., Virol., 147:217-222[1985]; and Schlehofer et al., Virol., 152:110-117 [1986]).

[0101] Alternatively, accessory functions can be provided using anaccessory function vector. Accessory function vectors include nucleotidesequences that provide one or more accessory functions. An accessoryfunction vector is capable of being introduced into a suitable host cellin order to support efficient AAV virion production in the host cell.Accessory function vectors can be in the form of a plasmid, phage,virus, transposon or cosmid. Accessory vectors can also be in the formof one or more linearized DNA or RNA fragments which, when associatedwith the appropriate control sequences and enzymes, can be transcribedor expressed in a host cell to provide accessory functions.

[0102] Nucleic acid sequences providing the accessory functions can beobtained from natural sources, such as from the genome of adenovirus, orconstructed using recombinant or synthetic methods known in the art. Inthis regard, adenovirus-derived accessory functions have been widelystudied, and a number of adenovirus genes involved in accessoryfunctions have been identified and partially characterized (See e.g.,Carter, “Adeno-Associated Virus Helper Functions,” in CRC Handbook ofParvoviruses, Vol. I (P. Tijssen, ed.) [1990], and Muzyczka, Curr. Top.Microbiol. Immun., 158:97-129 [1992]). Specifically, early adenoviralgene regions E1a, E2a, E4, VAI RNA and, possibly, E1b are thought toparticipate in the accessory process (Janik et al., Proc. Natl. Acad.Sci. USA 78:1925-1929 [1981]). Herpesvirus-derived accessory functionshave been described (See e.g., Young et al., Prog. Med. Virol., 25:113[1979]). Vaccinia virus-derived accessory functions have also beendescribed (See e.g., Carter, supra., and Schlehofer et al., Virol.,152:110-117 [1986]).

[0103] As a consequence of the infection of the host cell with a helpervirus, or transfection of the host cell with an accessory functionvector, accessory functions are expressed which transactivate the AAVhelper construct to produce AAV Rep and/or Cap proteins. The Repexpression products direct excision of the recombinant DNA (includingthe DNA of interest encoding at least a portion of Factor VIII) from theAAV vector. The Rep proteins also serve to duplicate the AAV genome. Theexpressed Cap proteins assemble into capsids, and the recombinant AAVgenome is packaged into the capsids. Thus, productive AAV replicationensues, and the DNA is packaged into rAAV virions.

[0104] Following recombinant AAV replication, rAAV virions can bepurified from the host cell using a variety of conventional purificationmethods, such as CsCl gradients. Further, if helper virus infection isemployed to express the accessory functions, residual helper virus canbe inactivated, using known methods. For example, adenovirus can beinactivated by heating to temperatures of approximately 60° C. forapproximately 20 minutes or more, as appropriate. This treatmentselectively inactivates the helper adenovirus which is heat labile,while preserving the rAAV which is heat stable.

[0105] III. Pharmaceutical Compositions

[0106] The resulting rAAV virions are then ready for use inpharmaceutical compositions which can be delivered to a subject, so asto allow production of biologically active Factor VIII. Pharmaceuticalcompositions comprise sufficient genetic material that allows therecipient to produce a therapeutically effective amount of Factor VIIIso as to reduce, stop and/or prevent hemorrhage. The compositions may beadministered alone or in combination with at least one other agent, suchas stabilizing compound, which may be administered in any sterile,biocompatible pharmaceutical carrier, including, but not limited to,saline, buffered saline, dextrose, and water. The compositions may beadministered to a patient alone, or in combination with other agents,clotting factors or factor precursors, drugs or hormones. In preferredembodiments, the pharmaceutical compositions also contain apharmaceutically acceptable excipient. Such excipients include anypharmaceutical agent that does not itself induce an immune responseharmful to the individual receiving the composition, and which may beadministered without undue toxicity. Pharmaceutically acceptableexcipients include, but are not limited to, liquids such as water,saline, glycerol, sugars and ethanol. Pharmaceutically acceptable saltscan be included therein, for example, mineral acid salts such ashydrochlorides, hydrobromides, phosphates, sulfates, and the like; andthe salts of organic acids such as acetates, propionates, malonates,benzoates, and the like. Additionally, auxiliary substances, such aswetting or emulsifying agents, pH buffering substances, and the like,may be present in such vehicles. A thorough discussion ofpharmaceutically acceptable excipients is available in Remington'sPharmaceutical Sciences (Mack Pub. Co., N.J. [1991]).

[0107] Pharmaceutical formulations suitable for parenteraladministration may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks's solution, Ringer'ssolution, or physiologically buffered saline. Aqueous injectionsuspensions may contain substances which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Additionally, suspensions of the active compounds may beprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles include fatty oils such as sesame oil, or syntheticfatty acid esters, such as ethyl oleate or triglycerides, or liposomes.Optionally, the suspension may also contain suitable stabilizers oragents which increase the solubility of the compounds to allow for thepreparation of highly concentrated solutions.

[0108] For topical or nasal administration, penetrants appropriate tothe particular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

[0109] The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art (e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes).

[0110] The pharmaceutical composition may be provided as a salt and canbe formed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at apH range of 4.5 to 5.5, that is combined with buffer prior to use.

[0111] After pharmaceutical compositions have been prepared, they can beplaced in an appropriate container and labeled for treatment. Foradministration of Factor VIII-containing vectors, such labeling wouldinclude amount, frequency, and method of administration.

[0112] Pharmaceutical compositions suitable for use in the inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. Determining atherapeutic effective dose is well within the capability of thoseskilled in the art using the techniques taught in the present invention,such as ELISA and ChromZ FVIII coagulation activity assay, and othertechniques known in the art. Therapeutic doses will depend on, amongother factors, the age and general condition of the subject, theseverity of hemophilia, and the strength of the control sequences. Thus,a therapeutically effective amount in humans will fall in a relativelybroad range that can be determined through clinical trials.

[0113] It is intended that the dosage treatment and regimen used withthe present invention will vary, depending upon the subject and thepreparation to be used. Thus, the dosage treatment may be a single doseschedule or a multiple dose schedule. Moreover, the subject may beadministered as many doses as appropriate to achieve or maintain thedesired blood clotting time.

[0114] Direct delivery of the pharmaceutical compositions in vivo willgenerally be accomplished via injection using a conventional syringe,although other delivery methods such as convention-enhanced delivery areenvisioned (See e.g., U.S. Pat. No. 5,720,720, incorporated herein byreference). In this regard, the compositions can be deliveredsubcutaneously, epidermally, intradermally, intrathecally,intraorbitally, intramucosally (e.g., nasally, rectally and vaginally),intraperitoneally, intravenously, intraarterially, orally, orintramuscularly. Other modes of administration include oral andpulmonary administration, suppositories, and transdermal applications.In particularly preferred embodiments, the compositions are administeredintravenously in the portal vasculature or hepatic artery

[0115] One skilled in the art will recognize that the methods andcompositions described above are also applicable to a range of othertreatment regimens known in the art. For example, the methods andcompositions of the present invention are compatible with ex vivotherapy (e.g., where cells are removed from the body, incubated with theAAV vector and the treated cells are returned to the body).

[0116] IV. Administration

[0117] AAV vector can be administered to any tissue suitable for theexpression of Factor VIII. In a preferred embodiments, the AAV vectorsof the present invention are successfully administered via the portalvasculature or hepatic artery where it is thought, without being boundby theory, that the vector transduces hepatocytes. Current approaches totargeting genes to the liver have focused upon ex vivo gene therapy. Exvivo liver-directed gene therapy involves the surgical removal of livercells, transduction of the liver cells in vitro (e.g., infection of theexplanted cells with recombinant retroviral vectors) followed byinjection of the genetically modified liver cells into the liver orspleen of the patient. A serious drawback for ex vivo gene therapy ofthe liver is the fact that hepatocyctes cannot be maintained andexpanded in culture. Therefore, the success of ex vivo liver-directedgene therapy depends upon the ability to efficiently and stably engraftthe genetically modified (i.e., transduced) hepatocytes and theirprogeny. It has been reported that even under optimal conditions,autologous modified liver cells injected into the liver or spleen whichengraft represent only a small percentage (less than 10%) of the totalnumber of cells in the liver. Ectopic engraftment of transduced primaryhepatocytes into the peritoneal cavity has been tried, in order toaddress the problem of engraftment in the liver.

[0118] Given the problems associated with ex vivo liver-directed genetherapy, in vivo approaches have been investigated for the transfer ofgenes into hepatocytes, including the use of recombinant retroviruses,recombinant adenoviruses, liposomes and molecular conjugates. Whilethese in vivo approaches do not suffer from the drawbacks associatedwith ex vivo liver-directed gene therapy, they do not provide a means tospecifically target hepatocytes. In addition, several of theseapproaches require performance of a partial hepatectomy, in order toachieve prolonged expression of the transferred genes in vivo.Adenovirus and molecular conjugate based delivery methods also result inliver toxicity and inflammation which is an undesirable feature ofFactor VIII gene therapy. The present invention provides compositionsand methods for the long-term expression of biologically active FactorVIII. It is contemplated that the present invention will bypass the needfor partial hepatectomy, while allowing expression of Factor VIII inconcentrations that are therapeutic in vivo. The present inventionfurther provides gene therapy compositions and methods that targethepatocytes for the production of Factor VIII by treated individuals.

[0119] Other tissues, however, may be suitable for the expression ofFactor VIII even if they are not the tissue that normally synthesizesthe protein. Muscle cells, for example, have been shown to expressbiologically active blood clotting Factor IX even though it is normallysynthesized in the liver.

[0120] Finally, the AAV vectors may contain any nucleic acid sequencescoding for biologically active Factor VIII. Additionally, the AAVvectors may contain a nucleic acid coding for fragments of Factor VIIIwhich is itself not biologically active, yet when administered into thesubject improves or restores the blood clotting time. For example, asdiscussed above, the Factor VIII protein comprises two polypeptidechains: a heavy chain and a light chain separated by a B-domain which iscleaved during processing. As demonstrated by the present invention,co-transducing recipient cells with the Factor VIII heavy and lightchains leads to the expression of biologically active Factor VIII.Because, however, most hemophiliacs contain a mutation or deletion inonly one of the chains (e.g., heavy or light chain), it may be possibleto administer only the chain defective in the patient and allow thepatient to supply the other chain. In this case, the AAV vector wouldfall within the scope of the invention even though the single chain(i.e., heavy or light) would not be biologically active until it wasadministered into a subject which can supply the second chain, thusforming biologically active Factor VIII.

[0121] V. Factor VIII Assays

[0122] As described in the Experimental section below, there are manyways to assay Factor VIII expression and activity. Although the presentinvention is not limited to immunoassay methods, the present inventionalso provides methods for detecting Factor VIII expression comprisingthe steps of: a) providing a sample suspected of containing Factor VIII,and a control containing a known amount of known Factor VIII; and b)comparing the test sample with the known control, to determine therelative concentration of Factor VIII in the sample. Thus, the methodsare capable of identifying samples (e.g., patient samples) withsufficient or insufficient quantities of Factor VIII. In addition, themethods may be conducted using any suitable means to determine therelative concentration of Factor VIII in the test and control samples,including but not limited to means selected from the group consisting ofWestern blot analysis, Northern blot analysis, Southern blot analysis,denaturing polyacrylamide gel electrophoresis (e.g., SDS-PAGE), reversetranscriptase-coupled polymerase chain reaction (RT-PCR), enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescentimmunoassay (IFA). Thus, the methods may be conducted to determine thepresence of normal Factor VIII sequences in the genome of the animalsource of the test sample, or the expression of Factor VIII (mRNA orprotein), as well as detect the presence of abnormal or mutated FactorVIII gene sequences in the test samples.

[0123] In one preferred embodiment, the presence of Factor VIII isdetected by immunochemical analysis. For example, the immunochemicalanalysis can comprise detecting binding of an antibody specific for anepitope of Factor VIII. In one another preferred embodiment of themethod, the antibody comprises polyclonal antibodies, while in anotherpreferred embodiment, the antibody comprises monoclonal antibodies.

[0124] It is further contemplated that antibodies directed against atleast a portion of Factor VIII will be used in methods known in the artrelating to the localization and structure of Factor VIII (e.g., forWestern blotting), measuring levels thereof in appropriate biologicalsamples, etc. The antibodies can be used to detect Factor VIII in abiological sample from an individual (e.g., an individual treated usingthe methods and/or compositions of the present invention). Thebiological sample can be a biological fluid, including, but not limitedto, blood, serum, plasma, interstitial fluid, urine, cerebrospinalfluid, synovial fluid, and the like. In particular, the antigen can bedetected from cellular sources, including, but not limited to,hepatocytes. For example, cells can be obtained from an individual andlysed (e.g., by freeze-thaw cycling, or treatment with a mild cytolyticdetergent including, but not limited to, TRITON X-100, digitonin,NONIDET P (NP)-40, saponin, and the like, or combinations thereof, See,e.g., International Patent Publication WO 92/08981).

[0125] The biological samples can then be tested directly for thepresence of the Factor VIII using an appropriate strategy (e.g., ELISAor RIA) and format (e.g., microwells, dipstick [e.g., as described inInternational Patent Publication WO 93/03367], etc.). Alternatively,proteins in the sample can be size separated (e.g., by polyacrylamidegel electrophoresis (PAGE), with or without sodium dodecyl sulfate(SDS), and the presence of Factor VIII detected by immunoblotting [e.g.,Western blotting]). Immunoblotting techniques are generally moreeffective with antibodies generated against a peptide corresponding toan epitope of a protein, and hence, are particularly suited to thepresent invention. In another preferred embodiment, the level of FactorVIII is assayed using the whole-blood clotting time and activated parialthromboplastin time (aPTT) of the subject's blood using techniques wellknown in the art (Herzog et al., Nature Medicine 5:56 [1999]).

[0126] The foregoing explanations of particular assay systems arepresented herein for purposes of illustration only, in fulfillment ofthe duty to present an enabling disclosure of the invention. It is to beunderstood that the present invention contemplates a variety ofimmunochemical assay protocols within its spirit and scope. Indeed,other methods such as biological assays to determine the presence andactivity of Factor VIII are also encompassed by the present invention.

[0127] Thus, in addition to the immunoassay systems described above,other assay systems, such as those designed to measure and/or detectFraction VIII and/or clotting ability of a subject's blood are alsoencompassed by the present invention (e.g., the ChromZ FVIII coagulationactivity [FVIII-c] assay [Helena Labs]).

Experimental

[0128] Below are examples of specific embodiments for carrying out thepresent invention. The examples are offered for illustrative purposesonly, and are not intended to limit the scope of the present inventionin any way.

[0129] Efforts have been made to ensure accuracy with respect to numbersused (e.g., amounts, temperatures, etc.), but some experimental errorand deviation should, of course, be allowed for.

[0130] In the experimental disclosure which follows, the followingabbreviations apply: N (Normal); M (Molar); mM (millimolar); μM(micromolar); g (grams); mg (milligrams); μg (micrograms); ng(nanograms); l or L (liters); ml (milliliters); μl (microliters); cm(centimeters); mm (millimeters); μm (micrometers); nm (nanometers); mU(milliunits); ⁵¹Cr (Chromium 51); μCi (microcurie); EC (degreesCentigrade); hFVIII (human factor VIII); FVIII (factor VIII); pH(hydrogen ion concentration); JRH grade; NaCl (sodium chloride); HCl(hydrochloric acid); OD (optical density); bp (base pair(s)); ATP(adenosine 5′-triphosphate); PCR (polymerase chain reaction); DNA(deoxyribonucleic acid); cDNA (complementary DNA); AAV (adeno-associatedvirus); rAAV (recombinant adeno-associated virus); ITR (invertedterminal repeat); FCS or FBS (fetal calf serum; fetal bovine serum); CFA(complete Freund's adjuvant); BSA (bovine serum albumin); ATCC (AmericanType Culture Collection, Rockville, Md.); Sigma (Sigma Aldrich, St.Louis, Mo.); Biodesign International (Biodesign International,Kennebunk, Mich.); Baxter Hyland (Baxter Healthcare Corp., BiotechGroup--Hyland Division, Hayward, Calif.); Helena Labs (HelenaLaboratories, Beaumont, Tex.); American Diagnostica (AmericanDiagnostica, Greenwich, Conn.); Accurate Chemical (Accurate Chemical andScientific Corp., Westbury, N.Y.); Molecular Probes (Molecular Probes,Eugene, Oreg.); Vysis (Vysis, Downer Grove, Ill.); Tel-Test (Tel-Test,Inc., Friendswood, Tex.); Molecular Dynamics (Molecular Dynamics,Sunnyvale, Calif.); NUNC (Naperville, Ill.); and Stratagene (StratageneCloning Systems, La Jolla, Calif.); Affinity Biologicals (AffinityBiologicals, Inc., Hamilton, Ontario); and Biodesign (BiodesignInternational, Kennebunkport, Me.).

EXAMPLE 1 Dual Vector Plasmid Construction

[0131] The heavy and light chains of human Factor VIII (hFVIII) wereassembled according to those reported by Yonemura et al (Yonemura etal., Prot. Engineer., 6:669-674 [1993]) and cloned as expressioncassettes into AAV vectors. Both vectors contain the promoter and thefirst non-coding intron (from −573 to +985) from the human elongationfactor 1α (EF1α) gene (Uetsuki et al, J. Biol. Chem., 264:5791-5798[1989]; and Kim et al., Gene 9:217-223 [1990]). Each vector alsocontains the first 57 base pairs of the FVIII heavy chain encoding the19 amino acid signal sequence. The heavy chain construct encodes the A1and A2 domains and 5 amino acids from the N terminus of the B domain.The light chain vector encodes 85 amino acids of the carboxy terminal Bdomain, in addition to the A3, C1, and C2 domains. Both vectors utilizethe human growth hormone (hGH) polyadenylation signal. The expressioncassettes were inserted between AAV ITRs. The initial cloning stepinvolved deleting 854 bp of EF1α sequences between the SpeI and XcmIsites of pVm4.1e-hFIX (Nakai et al., Blood 91:1-9 [1998]), andreligating to create pVm4.1eδD-hFIX.

[0132] This construct was then digested with EcoRI, which released thehFIX cDNA, and was ligated to an oligonucleotide containing MfeI ends(EcoRI-compatible) and an internal ClaI restriction site, creatingpVm4.1eδD-linker. The heavy and light chain fragments, including the hGHpolyadenylation sequences were isolated from pVm4.1cFVIII-HC andpVm4.1cFVIII-LC, respectively as ClaI-BstEII fragments. These fragmentswere cloned between the corresponding sites in the pVm4.1eδD-linker,creating plasmids pVm4.1eδD-FVIII-HC (also, rAAV-hFVIII-HC) andpVm4.1eδD-FVIII-LC (also, rAAV-hFVIII-LC).

[0133]FIG. 7 provides a map of the constructs. In this figure, the upperline in each panel represents the gene structure of the vectors, and thelower line represents the structure of the hFVIII protein domainsencoded by the vectors (ITR, AAV inverted terminal repeat; EF1αPro/Intron 1, human polypeptide elongation factor 1α gene promoter andfirst intron; hFVIII-HC human FVIII cDNA; HFVIII-LC, human FVIII cDNA;hGH PA, human growth hormone polyadenylation signal; SS, human FVIIIsignal sequence; A1, A2, “B”, A3, C1, C2, complete and incomplete (″)protein domains of the hFVIII protein).

EXAMPLE 2

[0134] Single Vector Plasmid Construction

[0135] The plasmid pAAV-F8-1 construct containing both the light andheavy chains of factor VIII was constructed as follows. A PCR fragment,Z8, containing cloning sites, 5 ′-splicing donor site of a syntheticintron based on EF1α and immunoglobulin G (IgG) intron sequences, Kozaksequence and the first 16 nucleotides of the human blood coagulationfactor VIII (FVIII) coding sequence was generated using oligonucleotidesZ8S and Z8A. The sequences of the nucleic acids is shown below:

[0136] Oligonucleotide Z8S:

[0137] 5′ cccaagcttgcggccgcccgggtgccgcccctaggcaggtaagtgccgtgtgtggttcc 3′(SEQ ID NO: 1)

[0138] Oligonucleotide Z8A:

[0139] 5′ ccgctcgagcagagctctatttgcatggtggaatcgatgccgcgggaaccacacacggc 3′(SEQ ID NO: 2)

[0140] PCR fragment Z8:

[0141] 5′cccaagcttgcggccgcccgggtgccgcccctaggcaggtaagtgccgtgtgtggttcccgcggcatcgattccaccatgcaaatagagctctgctcgagcgg 3′ (SEQ ID NO: 3)

[0142] Nucleic acid Z8 was inserted into pZERO-2 (Invitrogen) betweenHindIII and XhoI sites to create pZ8. A PCR fragment, INT3, containingthe branching point, the polypyrimidine tract, and the 3′ splicingacceptor site of the synthetic intron was generated usingoligonucleotides INT3S and INT3A whose sequence is shown below.

[0143] Oligonucleotide INT3S:

[0144] 5′ ttcccgcgggcctggcctctttacgggttatggcccttgcgtgccttgaattactga 3′(SEQ ID NO: 4)

[0145] Oligonucleotide INT3A:

[0146] 5′ gaatcgatacctgtggagaaaaagaaaaagtggatgtcagtgtcagtaattcaaggc 3′(SEQ ID NO: 5)

[0147] PCR Fragment INT3:

[0148]ccgcgggcctggcctctttacgggttatggcccttgcgtgccttgaattactgacactgacatccactttttctttttctccacaggtatcgattc 3′ (SEQ ID NO: 6)

[0149] 3 inserted between the SacII and ClaI sites of pZ8 to createpZ8.I. Therefore, Z8.I contains the entire synthetic intron betweenAvrII and ClaI sites. A hFVIII cDNA fragment having SacI and XhoIrestriction sites was inserted between the SacI and XhoI sites of pZ8.Ito create pZ8.I.dB. Therefore, pZ8.I.dB contains a synthetic intron andthe entire coding sequence of hFVIII.

[0150] A PCR fragment, EG3, containing three HNF-3 binding sites and −54to +8 of mouse albumin gene was generated using oligonucleotides EG3Sand EG3A with modifications to eliminate linker sequences. The sequencesof EG3S and EG3A are as follows:

[0151] Oligonucleotide EG3S:

[0152] 5 ′agggaatgtttgttcttaaataccatccagggaatgtttgttcttaaataccatccagggaatgtttgttcttaaataccatctacagttattggttaaa 3′ (SEQ ID NO: 7)

[0153] Oligonucleotide EG3A:

[0154] 5′ ggaaaggtgatctgtgtgcagaaagactcgctctaatatacttctttaaccaataactg 3′(SEQ ID NO: 8)

[0155] PCR Fragment EG3:

[0156]5′agggaatgtttgttcttaaataccatccagggaatgtttgttcttaaataccatccagggaatgtttgttcttaaataccatctacagttattggttaaagaagtatattagagcgagtctttctgcacacagatcacctttcc3′ (SEQ ID NO: 9)

[0157] EG3 was then phosphorylated using T4 polynucleotide kinase andinserted into the SmaI site of pZ8.I.dB to create pZ8.I.dB.egg. A DNAfragment, SPA, containing an efficient synthetic polyA signal based onrabbit β-globin sequences (Genes and Develop., 3:1019) was generated byhybridizing two oligonucleotides SPA.S and SPA.A.

[0158] Oligonucleotide SPA.S:

[0159] 5′ tcgagaataaaagatcagagctctagagatctgtgtgttggttttttgtgtgcggccgc 3′(SEQ ID NO: 10)

[0160] Oligonucleotide SPA.A:

[0161] 5 ′ tcgagcggccgcacacaaaaaaccaacacacagatctctagagctctgatcttttattc3′ (SEQ ID NO: 11)

[0162] PCR Fragment SPA:

[0163] 5 ′tcgagaataaaagatcagagctctagagatctgtgtgttggttttttgtgtgcggccgctcga 3′ (SEQID NO: 12)

[0164] SPA was inserted into the XhoI site of pZero-2 to createpZero-2.SPA. SPA was excised from a pZero-2.SPA clone and inserted intothe XhoI site of pZ8.I.dB.egg to create pZ8.I.dB.egg.A. pAAV-CMV-FIX9was digested with ClaI, blunted with T4 polymerase and religated tocreate pAAV(Cla⁻)-CMV-FIX9.

[0165] The entire expression cassette containing HNF-3.albuminpromoter-synthetic intron-hFVIII-synthetic poly A signal was excisedfrom pZ8.I.dB.egg.A using NotI and ligated to the plasmid backbone andAAV ITRs from pAAV (Cla-)-CMV-FIX9 to create pAAV-F8-1. The nucleotidesequence of the vector from ITR to ITR (i.e., excluding plasmidbackbone) is shown in SEQ ID NO 13.

EXAMPLE 3 Virion Production

[0166] AAV vectors were produced from these plasmids using the Ad freesystem as previously described in U.S. Pat. No. 5,858,351; U.S. Pat. No.5,846,528; U.S. Pat. No. 5,622,856; and Matsushita et al., Gene Ther5:938 (1998) all of which are hereby incorporated by reference. Briefly,293 cells (ATCC, catalog number CRL-1573) were seeded in 10 cm dishes ata density of 3×10⁶ cells per dish in 10 ml medium and incubated at 37°C. with CO₂ and humidity. After an overnight incubation, cells wereapproximately seventy to eighty percent confluent.

[0167] The cells were then transfected with DNA by the calcium phosphatemethod, as is well known in the art. Briefly, 7 μg of AAV vectorcontaining the Factor VIII coding region, 7 μg of pladeno5 whichsupplies the accessory functions, and 7 μg of 1909 AAV helper were addedto a 3 ml sterile, polystyrene snap cap tube using sterile pipette tips.Then, 1.0 ml of 300 mM CaCl₂ (JRH grade) was added to each tube andmixed by pipetting up and down. An equal volume of 2×HBS (274 mM NaCl,10 mM KCl, 42 mM HEPES, 1.4 mM Na₂PO₄, 12 mM dextrose, pH 7.05, JRHgrade) was added with a 2 ml pipette, and the solution was pipetted upand down three times. The DNA mixture was immediately added to thecells, one drop at a time, evenly throughout the 10 cm dish. The cellswere then incubated at 37° C. with CO₂ and humidity for six hours. Agranular precipitate was visible in the transfected cell cultures. Aftersix hours, the DNA mixture was removed from the cells, which wereprovided with fresh medium and incubated for 72 hours.

[0168] After 72 hours, the cells were harvested, pelleted, andresuspended in 1 ml TBS/1% BSA. Freeze/thaw extracts were prepared byrepeatedly (three times) freezing the cell suspension on dry ice andthawing at 37° C. Viral preps were stored at −80° C. and titered by dotblot assay prior to the first round of infection.

EXAMPLE 4 In Vitro Cell Transduction

[0169] Cells from the stable human cell line, 293 (ATCC No. CRL1573)were seeded in six-well plates (i.e., plates having six wells for cellgrowth) at a density of 5×10⁵ cells/well. When the monolayers reached80-90% confluence, they were infected with rAAV virionsAAV-eδD-FVIII-HC, AAV-eδD-FVIII-LC, an equal ratio of AAV-eδD-FVIII-HCand AAV-eδD-FVIII-LC, or AAV-eδD-FIX at MOIs of 3×10³ and 3×10⁴.Eighteen hours post infection, the media were replaced with DMEM/10%heat inactivated FBS. The media were collected later for analysis byELISA (as described below) for FVIII light chain antigen levels, and bythe ChromZ FVIII as coagulation activity (FVIII-c) assay (Helena Labs)for biological activity, using the manufacturer's instructions and asdescribed in Example 6.

EXAMPLE 5 Single Chain Factor VIII Infectivity Assay

[0170] In this Example, the infectivity of single chain Factor VIII wasinvestigated. To determine the infectivity of rAAV-hF8-1, HepG2, 293,and H2.35 cells were infected with rAAV-hF8-1 and a control vectorrAAV-hF8L at an MOI of 1×10⁴ viral particles per cell. Recombinant AAVDNA in infected cells was isolated by Hirt extraction and run on analkaline agarose gel. Southern blot analysis using an human F8 probeshowed that similar amounts of rAAV-hF8-1 and rAAV-hF8L were isolatedfrom uncoated virus in the infected cells. An infectious center assay(ICA) known in the art (See e.g., Snyder, Current Protocols in Genetics,Chapter 12, John Wiley & Sons [1997]) was used to further characterizethe infectivity of rAAV-hF8-1. In this assay, the infectious particle tototal particle ratio of rAAV-F8-1 and that of a control rAAV vector withthe genome size of 4645 nucleotides was determined. The resultsindicated that rAAV-hF8-1 had an infectious particle to total particleratio that was comparable to the control vector at approximately 1:1000.Taken together, these results indicate rAAV-hF8-1 has similarinfectivity as rAAV vectors with the genome size of wild-type AAV.

EXAMPLE 6 Factor VIII Protein Expression Assay

[0171] An ELISA specific for the light chain of FVIII was used todetermine FVIII light chain antigen levels in the 293 cells, as well asthe injected animals (described below). NUNC Maxisorb 96 well plateswere coated with 50 μl of a 1:500 dilution of the light chain specificantibody, N77110 (Biodesign International) in a coating buffer overnightat 4° C. The plate was washed three times with wash buffer (PBS, 0.05%Tween 20) and blocked with 200 μl blocking buffer (PBS, 10% horse serum,0.05% Tween 20) at room temperature for 1 hour. The plate was washedthree times and standards and samples were applied. Bioclate recombinanthuman FVIII (Baxter Hyland) was used as the standard, and was diluted inblocking buffer to concentrations ranging from 320 ng/ml to 10 ng/ml.

[0172] For analysis of transduced culture supernatants, the standardscontained 50% media, and for analysis of mouse plasma, the standardswere diluted into 10%, in normal pooled mouse plasma (Sigma). A standardassay reference plasma (SARP; Helena Labs) was also included in theassay. Following the loading of the standards and samples (95 μl/well),the plate was incubated at room temperature for 2 hours, and washed fivetimes with wash buffer (200 μl/well). A 1:200 dilution of a horseradishperoxidase-conjugated light chain specific antibody, ESH8-HRP, (AmericanDiagnostica) was added (100 μl/well), and the plate was incubated for 1hour at room temperature. The plates were then washed four times withwash buffer, and the antigen was detected using an ABTS peroxidasesubstrate kit (BioRad) according to the manufacturer's instructions. Theresults are shown in Table 1 of Example 7, below.

EXAMPLE 7 Factor VIII Biological Activity Assay

[0173] The ChromZ FVIII coagulation activity (FVIII-c) assay (HelenaLabs, Beaumont, Tex.) was used to detect biologically active FVIII inthe 293 cells infected as described in Example 4. Bioclate recombinanthuman FVIII (Baxter Hyland) was used as a standard to analyzetransfected culture supernatants. The standards were diluted in plasmadilution buffer (supplied in kit) in the range of 10 ng/ml to 0.313ng/ml, and were made 2.5% in media. Because this assay can detect bothhuman and murine FVIII activity, it was modified to deplete biologicallyactive human Factor VIII in the mouse plasma. Mouse plasma waspre-incubated with an antibody specific for human FVIII prior toperforming the assay. The difference in FVIII activity between theuntreated plasma sample and the antibody treated sample represent theamount of biologically active human FVIII in the plasma. The standardused in the assay was normal pooled human plasma (FACT; obtained fromGeorge King Biomedical). Serial dilutions of FACT were made in FVIIIdeficient plasma from undiluted (200 ng/ml) to 6.25 ng/ml. The standards(10 μl) were incubated at 37° C. for 15 min., with or without theaddition of 2 μl antibody N77110. Similarly, mouse plasma samples werediluted in FVIII deficient plasma and 10 μl of these diluted sampleswere incubated with or without 2 μl of N77110 at 37° C. for 15 min., andimmediately placed on ice. Thus, all incubations with antibody were donein a background of 100% plasma. The antibody adsorbed and non-adsorbedFACT standards, as well as the mouse plasma samples were diluted 1:20 inplasma detection buffer provided in the ChromZ kit. Thus, the finalconcentration of the FACT standards used in the assay ranged from 10ng/ml to 0.313 ng/ml.

[0174] Twenty five microliters of these dilutions were added to achilled 96 well plate. With the plate on ice, 25 μl of FIXa reagent and50 μl of FX were added, and the plate was incubated at 37° C. for 15min. Substrate (50 μl) was added and the plate was incubated for anadditional 3 min at 37° C. The reaction was stopped with the addition of25 μl 50% acetic acid and the optical density at 405 nm was measured.

[0175] As shown below in Table 1, infection of 293 cells withAAV-eδD-FVIII-HC resulted in no antigen production, as well as nobiologically-active protein. Cells infected with AAV-eδD-FVIII-LCproduced FVIII light chain, but no biologically active protein. However,cells transduced with both vectors produced FVIII light chain andbiologically active FVIII in a dose-dependent manner. Transduction ofcells with the negative control vector, AAV-eδD-FIX, resulted in noantigen nor any biologically active FVIII. It was assumed that equalamounts of heavy and light chains were produced in transduced cells. Theactivity units were converted to nanograms using the definition of oneunit being equal to the amount of FVIII in 1 ml of normal pooled humanplasma, or 200 ng. TABLE 1 In Vitro Production of Biologically ActiveHuman Factor VIII From Two rAAV Vectors ELISA ChromZ Vector MOI (ng/ml)(mU/ml) (ng/ml) AAV-eδD-FVIII-HC and 3 × 10³ 24 35 7.1 AAV-eδD-FVIII-LCAAV-eδD-FVIII-HC and 3 × 10⁴ 121 440 87.9 AAV-eδD-FVIII-LCAAV-eδD-FVIII-HC 3 × 10³ 0 0 0 AAV-eδD-FVIII-HC 3 × 10⁴ 0 0 0AAV-eδD-FVIII-LC 3 × 10³ 20.5 0 0 AAV-eδD-FVIII-LC 3 × 10⁴ 96.9 0 0AAV-hFIX 3 × 10³ 0 0 0 AAV-hFIX 3 × 10⁴ 0 0 0 No Vector 0 0 0

EXAMPLE 8 Immunofluorescent Staining of FVIII Heavy and Light Chains

[0176] In these experiments, 293 cells transduced as described abovewere analyzed using immunofluorescent staining. 293 cells were plated onrat tail collagen-coated two-well culture slides at a density of 4×10⁵cells per well. Forty-eight hours later, the cells were transduced at anMOI of 3×10⁴ particles per cell of rAAV-hFVIII-HC and rAAV-hFVIII-LC.Forty-eight hours post-transduction, the cells were fixed in situ withacetone, blocked with 2% BSA, and stained with a fluorescently labelledanti-hFVIII light chain antibody and a fluorescently labelledanti-hFVIII heavy chain antibody. The anti-hFVIII light chain antibodyused was ESH-4 monoclonal antibody (American Diagnostica), fluorescentlylabelled with alexa-488 (Molecular Probes), according to themanufacturer's instructions. The anti-hFVIII heavy chain antibody usedwas MAS530P monoclonal antibody (Accurate Chemical) fluorescentlylabelled with alexa-594 (Molecular Probes), according to themanufacturer's instructions. The cells were counter-stained with DAPI.The images were collected using a Zeiss Axioskop fluorescence microscopeequipped with separate filters for DAPI, FITC, and rhodamine signals anda CCD camera. Image analysis was performed using Quips imaging software(Vysis).

[0177] As indicated above, infection of cells with eitherrAAV-eδD-FVIII-HC or AAV-eδD-FVIII-LC, followed by staining withantibodies to both chains resulted in production of the individualchains of human FVIII. Immunofluorescent staining of cells co-infectedwith both vectors demonstrated that although some cells express only theheavy or light chain of hFVIII, many co-expressed both chains of humanFVIII.

EXAMPLE 9 In Vitro Expression of Factor VIII Using Single Construct

[0178] Table 2 shows that two single vector constructs containing theheavy and the light chain of Factor VIII driven by different promotersexpress biologically active Factor VIII. The constructs pAAV-hF8-1 (SEQID NO: 13), and pVm4.1cF8ΔB (SEQ ID NO: 14) were transfected into 293cells. Following transfection, the cells were allowed to express factorVIII for 48-72 hours. Factor VIII in the culture media was assayed bythe ChromZ FVIII coagulation activity (FVIII-c) assay, as per themanufacturer's instructions. TABLE 2 In Vitro Production of BiologicallyActive FVIII ELISA ChromZ Construct(s) (ng/ml) (ng/ml) Control — 0pAAV-hF8-1 — 4.9 pVm4.1cF8ΔB — 46

EXAMPLE 10 Factor VIII Expression Using Tissue Specific Promoters

[0179] In these experiments, different promoters and enhancer elementswere used to drive expression of a Factor VII coding sequence.Expression of Factor VIII was compared in 293 cells and HepG2 cellsusing different promoters. The pAAVeF8ΔB contains an EF-1α promoter witha hGH intron, Factor VIII with a B-domain deletion (F8ΔB) and a polyA.As described previously, pAAV-hF8-1 uses the HNF-3 albumin promoter witha minimal intron followed by F8ΔB and a minimal polyA. The constructpAAV-c8 uses the CMV enhancer-promoter and the F8ΔB. pAAV8b1 containsthe HNF-3 albumin promoter followed by the CMV/B-globin intron with theF8ΔB and a minimal poly A site. Table 3 describes Factor VIII expressionusing the albumin promoter relative to the control plasmid pV4.1 eF8ΔBin HepG2 and 293 cells. These data show increased expression of FactorVIII in HepG2 liver cells with the albumin promoter as compared toFactor VIII expression in 293 cells. TABLE 3 Relative Tissue Specificityof Promoters Plasmid Construct HepG2 Cells 293 Cells pAAV-hF8-1 6.2 0.6pAAV8b1 6.7 1.0 pAAVc8 30.0 41.0 pV4.1eF8ΔB 100 100

[0180] Next, several promoters derived from the transthyretin (TTR) genepromoter were transfected into HepG2 cells. TTR is an abundant serumprotein and the gene enhancer-promoter contains well knownliver-specific transcription factor binding sites (Samadani et al., GeneExpression 6:23 [1996]; Yan et al., EMBO 9:869 [1990]; Costa andGrayson, Nuc. Acids Res., 19:4139 [1991]; Costa et al., Mol. Cell. Bio.,6:4697 [1986]). The constructs were made by replacing the HNF-3 albuminpromoter in pAAV-hF8-1 with various lengths of the TTRpromoter-enhancer. The TTR enhancer-promoter was modified by replacingthe weak affinity binding sites with the strong affinity binding sitesto create pAAV-hF8-2. The pAAV-hF8-TTR-E-L-P202 construct contains thefull TTR promoter with a linker between the enhancer and the promoter.The remaining constructs are 5′ deletions: pAAV-hF8-TTR-E-P202 has thepromoter and enhancer with no linker; pAAV-hF8-TTR-E-P197 has a 5 basepair deletion from the promoter; pAAV-hF8-TTR-E-P151 has a 50 base pairdeletion; pAAV-hF8-TTR-P202 lacks the TTR enhancer and pAAV-hF8-TTR(X)has a 65 base pair deletion in the enhancer. The control plasmid,pAAV-hF8-1, expressed approximately 4.6 mU/ml. Table 4 shows thefold-increase in Factor VIII activity using the TTR promoter seriesrelative to the control plasmid. TABLE 4 Factor VIII Expression UsingTTR-Derived Promoters Relative Factor Plasmid Construct VIII ActivitypAAV-hF8-STTR 3.16 pAAV-hF8-TTR-E-L-P202 8.86 pAAV-hF8-TTR-E-P202 6.1pAAV-hF8-TTR-EP197 7.3 pAAV-hF8-TTR-E-P151 13.3 pAAV-hF8-TTR-P202 2.3

EXAMPLE 11 In Vivo Expression of Factor VIII

[0181] In order to test the feasibility of the AAV vector approach ofthe present invention in vivo, three groups of five C57BL/6 mice wereinjected via the portal vein with either 3×10¹¹ particles ofAAV-eδD-FVIII-HC, 3×10¹¹ particles of AAV-eδD-FVIII-LC, or 3×10¹¹particles of both AAV-eδD-FVIII-HC and AAV-eδD-FVIII-LC. In addition, agroup of four animals was injected with 3×10¹¹ particles of AAV-eδD-FIX.It has been shown that this strain of mice does not elicit an immuneresponse to human FVIII when the gene is delivered to the liver via anadenoviral vector (Connelly et al., Blood 87:4671-4677 [1996]). Asindicated by the results shown below, the data obtained during theseexperiments demonstrate the feasibility of producing biologically activeFVIII using two AAV vectors to independently deliver the heavy and lightchains of FVIII.

[0182] Blood samples were collected in sodium citrate via theretro-orbital plexus at biweekly intervals for the first 2 months and atmonthly intervals thereafter for 6 months and at 11 months. Very highlevels of FVIII light chain were expressed in animals injected withAAV-eδD-FVIII-LC alone or both vectors as shown in FIG. 8.

[0183] In order to assess the amount of biologically active human FVIIIproduced in the animals, a modified ChromZ assay was used. Since thisassay detects both human and murine FVIII, the amount of FVIII presentin- the plasma before and after adsorption to an antibody specific tohuman FVIII was determined. The amount of FVIII remaining in the plasmaafter adsorption represented the amount of active murine FVIII and thedifference represented the amount of active human FVIII. Controlexperiments demonstrated that the antibody could remove 80-90% of thehuman FVIII from a mouse plasma sample when the sample was spiked withup to 32 ng of human FVIII. The modified ChromZ assay indicated thatonly those animals injected with both vectors produced biologicallyactive FVIII, as indicated in Table 5. The results shown in Table 5 arethose from plasma collected 8 weeks post-injection, although similarresults were obtained at 10 weeks and 5 months post-injection. One ofthe five animals co-injected with both the heavy and light chain vectordid not express VIII, presumably due to an inefficient injection, andwas omitted from the analysis. Animals injected with both vectorsproduced over 2 μg/ml hFVIII light chain as measured by ELISA. TheChromZ assay indicated that a total of 600-900 ng/ml of active hFVIIIwas detected in the plasma. The contribution from murine Factor VIII wasapproximately 400-500 ng/ml, indicating that about 230-430 ng/ml ofactive human Factor VIII was present in the plasma. Although only afraction of the total protein was found to be active, the animalsproduced physiological levels of the active protein (i.e., 200 ng/ml).The animals were found to have maintained these physiological levels ofactive protein for more than 11 months, without waning. Similar analysesperformed on animals injected with the light chain vector alone, theheavy chain vector alone, or the HFIX vector demonstrated nobiologically active human FVIII in the plasma of these animals. TABLE 5Biological Activity of Human Factor VIII In Vivo Total FVIII MurineHuman ELISA (−Ab) FVIII (+Ab) FVIII Construct(s) Used (ng/ml) (Units)(Units) (ng/ml) AAV-eδD-FVIII-HC 2288 3.9 2.2 342 and AAV-eδD-FVIII-LC*AAV-eδD-FVIII-LC* 3329 1.4 1.6 0 AAV-eδD-FVIII-HC 0 1.6 1.6 0AAV-eδD-FIX 0 1.4 2.0 0

EXAMPLE 12 Gene Transfer and Vector Expression in Tissues

[0184] In these experiments, evidence of gene transfer to liver wasobtained by Southern Blot analysis following isolation of DNA from oneanimal of each experimental group sacrificed 8 weeks post-injection(i.e., as described in Example 11). In addition, DNA was obtained fromother tissues in order to determine the degree of vector expression inorgans other than the liver.

[0185] Twenty micrograms of DNA was digested with BglII, separated usinga 1% agarose gel, and hybridized with a ³²P-labelled 1126 bp AlwNIfragment encoding the A1 and A2 domains of hFVIII (heavy chain probe),or a ³²P-labelled 1456 bp NdeI-EcoRI fragment encoding the A3, C1 an C2domains of hFVIII (light chain probe). Copy number controls weregenerated by spiking Bg/II-digested naive mouse liver DNA withBlgII-digested heavy or light chain plasmid DNA (pVm4.1eδD-hFVIII-HC andpVm4.1eδD-hFVIII-LC, respectively), at ratios of 10, 5, 1, 01, and 0.01copies per diploid genome. The hybridized membranes were analyzed usinga Storm 860 phosphoimager (Molecular Dynamics), and quantitation ofvector copy number was evaluated using ImageQuaNT software (MolecularDynamics). Autoradiography of the hybridized membranes was alsoperformed. Total RNA was isolated from liver tissue using the RNA Statextraction kit (Tel-Test). As describe briefly below, Northern blotanalysis was also performed on 10 μg RNA using methods known in the art,in conjunction with the ³²P-labelled probes specific to the heavy andlight chains of hFVIII described above and autoradiography was performedon the hybridized membranes.

[0186] Following digestion of liver DNA with BglII and hybridizationwith an hFVIII light chain probe described below, using methods known inthe art, a band at the predicted size of 3015 bp was detected in animalsinjected with rAAV-hFVIII-LC, or both the heavy and light chain vectors.This band was not observed in the DNA of animals injected with the heavychain vector alone or the hFIX vector, as shown in FIG. 9A(rAAV-hFVIII-LC, lane 1; rAAV-hFIX, lane 2; rAAV-hFVIII-HC, lane 3; bothrAAV-hFVIII-LC and rAAV-hFVIII-HC, lane 4; copy number controls weregenerated by spiking BglII digested naive mouse liver DNA with thecorresponding plasmids at ratios of 10, 5, 1, 0.1, and 0.01 copies perdiploid genome, lanes 5-9). Phosphoimage analysis revealed that thelight chain vector was present at approximately 2.4 and 1.5 copies perdiploid genome in animals injected with the light chain vector alone orboth vectors, respectively. When BglII-digested DNA was hybridized withan hFVIII heavy chain probe, the expected band of 2318 bp was observedin animals injected with the heavy chain vector alone or both vectors,but was not detected in animals injected with the light chain vectoralone or the hFIX vector, as shown in FIG. 9B. The copy number inanimals injected with the heavy chain vector alone and both vectors was1.1 and 1.7 vector copies per diploid genome, respectively.

[0187] The results of hybridization of DNA extracted from the spleen,kidney and heart tissue with either an hFVIII light chain probe or aheavy chain probe indicated that these tissues contained less than 1copy of vector sequences per 10 diploid genomes, demonstrating that thevector distributes primarily to the liver following intra-portalinjection, as shown in FIGS. 10A and 10B.

[0188] Human FVIII gene expression in the liver of the mice was alsoassessed by Northern blot analysis on RNA isolated from animalssacrificed 8 weeks post-injection (as described above). hFVIII lightchain transcripts of the predicted size (2.7 kb) were observed inanimals injected with the light chain vector alone or both vectors, asshown in FIG. 11A. Similarly, the expected hFVIII heavy chaintranscripts (2.7 kb) were detected in animals that were injected withthe heavy chain vector alone or both vectors, as shown in FIG. 11B.Since the heavy and light chain DNA sequences were shown by Southernblot analysis to be present at approximately the same copy number (1.7and 1.5 copies per diploid genome, respectively), in an animal injectedwith both vectors, these results demonstrate that both the heavy andlight chains of hFVIII are expressed in the liver in approximatelyequivalent amounts.

EXAMPLE 13 In vivo Expression of Factor VIII Using Single Vectors

[0189] Several groups comprising four C57BL/6 mice were injected via theportal vein with 3×10¹¹ particles of AAV-hF8-STTR, 3×10¹¹ particles ofAAV-hF8-hAAT-137, or 3×10¹¹ particles of AAV-hF8-HNF3-alb-original. Asdiscussed above, AAV-hF8-STTR contains modified sequences from thetransthyretin gene promoter. AAV-hF8-hAAT-137 contains 137 base pairs ofthe human,-antitrypsin promoter. See De Simone et al., EMBO J.,6:2759-2766 (1987). AAV-hF8-HNF3-alb-original, like AAV-F8-1, containsthree HNF3 binding sites and 54 base pairs of the albumin promoter.

[0190] Expression of Factor VIII was measured at 4 weeks by human FactorVIII-specific ELISA (Affinity Biologicals). Even at this earlytime-point, several of these mice expressed between 2 and 20 ng/ml ofhuman Factor VIII. Table 6 shows expression levels at 4 weekspost-infection for selected animals. TABLE 6 Factor VIII Expression at 4Weeks rAAV Factor VIII Levels (ng/ml) rAAV-hF8-STTR (mouse #1) 4.3rAAV-hF8-STTR (mouse #2) 2.4 rAAV-hF8-hAAT-137 (mouse #1) 10.5rAAV-hF8-hAAT-137 (mouse #2) 20.4 rAAV-hF8-HNF3-alb-original 2.2 (mouse#1)

[0191] rAAV-hF8-hAAT-137 produced expression levels of as much as 10 to20 ng/ml, which represent 20% to 40% of normal human levels of FactorVIII. These data therefore show the in vivo expression oftherapeutically effective amounts of human Factor VIII using

1 15 1 59 DNA Artificial Sequence Oligonucleotide Z8S 1 cccaagcttgcggccgcccg ggtgccgccc ctaggcaggt aagtgccgtg tgtggttcc 59 2 59 DNAArtificial Sequence Oligonucleotide Z8A 2 ccgctcgagc agagctctatttgcatggtg gaatcgatgc cgcgggaacc acacacggc 59 3 103 DNA ArtificialSequence PCR fragment Z8 3 cccaagcttg cggccgcccg ggtgccgccc ctaggcaggtaagtgccgtg tgtggttccc 60 gcggcatcga ttccaccatg caaatagagc tctgctcgag cgg103 4 57 DNA Artificial Sequence Oligonucleotide INT3S 4 ttcccgcgggcctggcctct ttacgggtta tggcccttgc gtgccttgaa ttactga 57 5 57 DNAArtificial Sequence Oligonucleotide INT3A 5 gaatcgatac ctgtggagaaaaagaaaaag tggatgtcag tgtcagtaat tcaaggc 57 6 99 DNA Artificial SequencePCR fragment INT3 6 ttcccgcggg cctggcctct ttacgggtta tggcccttgcgtgccttgaa ttactgacac 60 tgacatccac tttttctttt tctccacagg tatcgattc 99 7100 DNA Artificial Sequence Oligonucleotide EG3S 7 agggaatgtt tgttcttaaataccatccag ggaatgtttg ttcttaaata ccatccaggg 60 aatgtttgtt cttaaataccatctacagtt attggttaaa 100 8 59 DNA Artificial Sequence OligonucleotideEG3A 8 ggaaaggtga tctgtgtgca gaaagactcg ctctaatata cttctttaac caataactg59 9 144 DNA Artificial Sequence PCR fragment EG3 9 agggaatgtttgttcttaaa taccatccag ggaatgtttg ttcttaaata ccatccaggg 60 aatgtttgttcttaaatacc atctacagtt attggttaaa gaagtatatt agagcgagtc 120 tttctgcacacagatcacct ttcc 144 10 59 DNA Artificial Sequence Oligonucleotide SPA.S10 tcgagaataa aagatcagag ctctagagat ctgtgtgttg gttttttgtg tgcggccgc 5911 59 DNA Artificial Sequence Oligonucleotide SPA.A 11 tcgagcggccgcacacaaaa aaccaacaca cagatctcta gagctctgat cttttattc 59 12 63 DNAArtificial Sequence PCR fragment SPA 12 tcgagaataa aagatcagag ctctagagatctgtgtgttg gttttttgtg tgcggccgct 60 cga 63 13 11933 DNA ArtificialSequence Vector from ITR to ITR 13 cagctgcgcg ctcgctcgct cactgaggccgcccgggcaa agcccgggcg tcgggcgacc 60 tttggtcgcc cggcctcagt gagcgagcgagcgcgcagag agggagtggc caactccatc 120 actaggggtt cctgcggccg cccagggaatgtttgttctt aaataccatc cagggaatgt 180 ttgttcttaa ataccatcca gggaatgtttgttcttaaat accatctaca gttattggtt 240 aaagaagtat attagagcga gtctttctgcacacagatca cctttccggg tgccgcccct 300 aggcaggtaa gtgccgtgtg tggttcccgcgggcctggcc tctttacggg ttatggccct 360 tgcgtgcctt gaattactga cactgacatccactttttct ttttctccac aggtatcgat 420 tccaccatgc aaatagagct ctccacctgcttctttctgt gccttttgcg attctgcttt 480 agtgccacca gaagatacta cctgggtgcagtggaactgt catgggacta tatgcaaagt 540 gatctcggtg agctgcctgt ggacgcaagatttcctccta gagtgccaaa atcttttcca 600 ttcaacacct cagtcgtgta caaaaagactctgtttgtag aattcacgga tcaccttttc 660 aacatcgcta agccaaggcc accctggatgggtctgctag gtcctaccat ccaggctgag 720 gtttatgata cagtggtcat tacacttaagaacatggctt cccatcctgt cagtcttcat 780 gctgttggtg tatcctactg gaaagcttctgagggagctg aatatgatga tcagaccagt 840 caaagggaga aagaagatga taaagtcttccctggtggaa gccatacata tgtctggcag 900 gtcctgaaag agaatggtcc aatggcctctgacccactgt gccttaccta ctcatatctt 960 tctcatgtgg acctggtaaa agacttgaattcaggcctca ttggagccct actagtatgt 1020 agagaaggga gtctggccaa ggaaaagacacagaccttgc acaaatttat actacttttt 1080 gctgtatttg atgaagggaa aagttggcactcagaaacaa agaactcctt gatgcaggat 1140 agggatgctg catctgctcg ggcctggcctaaaatgcaca cagtcaatgg ttatgtaaac 1200 aggtctctgc caggtctgat tggatgccacaggaaatcag tctattggca tgtgattgga 1260 atgggcacca ctcctgaagt gcactcaatattcctcgaag gtcacacatt tcttgtgagg 1320 aaccatcgcc aggcgtcctt ggaaatctcgccaataactt tccttactgc tcaaacactc 1380 ttgatggacc ttggacagtt tctactgttttgtcatatct cttcccacca acatgatggc 1440 atggaagctt atgtcaaagt agacagctgtccagaggaac cccaactacg aatgaaaaat 1500 aatgaagaag cggaagacta tgatgatgatcttactgatt ctgaaatgga tgtggtcagg 1560 tttgatgatg acaactctcc ttcctttatccaaattcgct cagttgccaa gaagcatcct 1620 aaaacttggg tacattacat tgctgctgaagaggaggact gggactatgc tcccttagtc 1680 ctcgcccccg atgacagaag ttataaaagtcaatatttga acaatggccc tcagcggatt 1740 ggtaggaagt acaaaaaagt ccgatttatggcatacacag atgaaacctt taagactcgt 1800 gaagctattc agcatgaatc aggaatcttgggacctttac tttatgggga agttggagac 1860 acactgttga ttatatttaa gaatcaagcaagcagaccat ataacatcta ccctcacgga 1920 atcactgatg tccgtccttt gtattcaaggagattaccaa aaggtgtaaa acatttgaag 1980 gattttccaa ttctgccagg agaaatattcaaatataaat ggacagtgac tgtagaagat 2040 gggccaacta aatcagatcc tcggtgcctgacccgctatt actctagttt cgttaatatg 2100 gagagagatc tagcttcagg actcattggccctctcctca tctgctacaa agaatctgta 2160 gatcaaagag gaaaccagat aatgtcagacaagaggaatg tcatcctgtt ttctgtattt 2220 gatgagaacc gaagctggta cctcacagagaatatacaac gctttctccc caatccagct 2280 ggagtgcagc ttgaggatcc agagttccaagcctccaaca tcatgcacag catcaatggc 2340 tatgtttttg atagtttgca gttgtcagtttgtttgcatg aggtggcata ctggtacatt 2400 ctaagcattg gagcacagac tgacttcctttctgtcttct tctctggata taccttcaaa 2460 cacaaaatgg tctatgaaga cacactcaccctattcccat tctcaggaga aactgtcttc 2520 atgtcgatgg aaaacccagg tctatggattctggggtgcc acaactcaga ctttcggaac 2580 agaggcatga ccgccttact gaaggtttctagttgtgaca agaacactgg tgattattac 2640 gaggacagtt atgaagatat ttcagcatacttgctgagta aaaacaatgc cattgaacca 2700 agaagcttcg aaataactcg tactactcttcagtcagatc aagaggaaat tgactatgat 2760 gataccatat cagttgaaat gaagaaggaagattttgaca tttatgatga ggatgaaaat 2820 cagagccccc gcagctttca aaagaaaacacgacactatt ttattgctgc agtggagagg 2880 ctctgggatt atgggatgag tagctccccacatgttctaa gaaacagggc tcagagtggc 2940 agtgtccctc agttcaagaa agttgttttccaggaattta ctgatggctc ctttactcag 3000 cccttatacc gtggagaact aaatgaacatttgggactcc tggggccata tataagagca 3060 gaagttgaag ataatatcat ggtaactttcagaaatcagg cctctcgtcc ctattccttc 3120 tattctagcc ttatttctta tgaggaagatcagaggcaag gagcagaacc tagaaaaaac 3180 tttgtcaagc ctaatgaaac caaaacttacttttggaaag tgcaacatca tatggcaccc 3240 actaaagatg agtttgactg caaagcctgggcttatttct ctgatgttga cctggaaaaa 3300 gatgtgcact caggcctgat tggaccccttctggtctgcc acactaacac actgaaccct 3360 gctcatggga gacaagtgac agtacaggaatttgctctgt ttttcaccat ctttgatgag 3420 accaaaagct ggtacttcac tgaaaatatggaaagaaact gcagggctcc ctgcaatatc 3480 cagatggaag atcccacttt taaagagaattatcgcttcc atgcaatcaa tggctacata 3540 atggatacac tacctggctt agtaatggctcaggatcaaa ggattcgatg gtatctgctc 3600 agcatgggca gcaatgaaaa catccattctattcatttca gtggacatgt gttcactgta 3660 cgaaaaaaag aggagtataa aatggcactgtacaatctct atccaggtgt ttttgagaca 3720 gtggaaatgt taccatccaa agctggaatttggcgggtgg aatgccttat tggcgagcat 3780 ctacatgctg ggatgagcac actttttctggtgtacagca ataagtgtca gactcccctg 3840 ggaatggctt ctggacacat tagagattttcagattacag cttcaggaca atatggacag 3900 tgggccccaa agctggccag acttcattattccggatcaa tcaatgcctg gagcaccaag 3960 gagccctttt cttggatcaa ggtggatctgttggcaccaa tgattattca cggcatcaag 4020 acccagggtg cccgtcagaa gttctccagcctctacatct ctcagtttat catcatgtat 4080 agtcttgatg ggaagaagtg gcagacttatcgaggaaatt ccactggaac cttaatggtc 4140 ttctttggca atgtggattc atctgggataaaacacaata tttttaaccc tccaattatt 4200 gctcgataca tccgtttgca cccaactcattatagcattc gcagcactct tcgcatggag 4260 ttgatgggct gtgatttaaa tagttgcagcatgccattgg gaatggagag taaagcaata 4320 tcagatgcac agattactgc ttcatcctactttaccaata tgtttgccac ctggtctcct 4380 tcaaaagctc gacttcacct ccaagggaggagtaatgcct ggagacctca ggtgaataat 4440 ccaaaagagt ggctgcaagt ggacttccagaagacaatga aagtcacagg agtaactact 4500 cagggagtaa aatctctgct taccagcatgtatgtgaagg agttcctcat ctccagcagt 4560 caagatggcc atcagtggac tctcttttttcagaatggca aagtaaaggt ttttcaggga 4620 aatcaagact ccttcacacc tgtggtgaactctctagacc caccgttact gactcgctac 4680 cttcgaattc acccccagag ttgggtgcaccagattgccc tgaggatgga ggttctgggc 4740 tgcgaggcac aggacctcta ctgactcgagaataaaagat cagagctcta gagatctgtg 4800 tgttggtttt ttgtgtgcgg ccgcaggaacccctagtgat ggagttggcc actccctctc 4860 tgcgcgctcg ctcgctcact gaggccgggcgaccaaaggt cgcccgacgc ccgggctttg 4920 cccgggcggc ctcagtgagc gagcgagcgcgcagctgcct gcaggacatg tgagcaaaag 4980 gccagcaaaa ggccaggaac cgtaaaaaggccgcgttgct ggcgtttttc cataggctcc 5040 gcccccctga cgagcatcac aaaaatcgacgctcaagtca gaggtggcga aacccgacag 5100 gactataaag ataccaggcg tttccccctggaagctccct cgtgcgctct cctgttccga 5160 ccctgccgct taccggatac ctgtccgcctttctcccttc gggaagcgtg gcgctttctc 5220 atagctcacg ctgtaggtat ctcagttcggtgtaggtcgt tcgctccaag ctgggctgtg 5280 tgcacgaacc ccccgttcag cccgaccgctgcgccttatc cggtaactat cgtcttgagt 5340 ccaacccggt aagacacgac ttatcgccactggcagcagc cactggtaac aggattagca 5400 gagcgaggta tgtaggcggt gctacagagttcttgaagtg gtggcctaac tacggctaca 5460 ctagaaggac agtatttggt atctgcgctctgctgaagcc agttaccttc ggaaaaagag 5520 ttggtagctc ttgatccggc aaacaaaccaccgctggtag cggtggtttt tttgtttgca 5580 agcagcagat tacgcgcaga aaaaaaggatctcaagaaga tcctttgatc ttttctacgg 5640 ggtctgacgc tcagtggaac gaaaactcacgttaagggat tttggtcatg agattatcaa 5700 aaaggatctt cacctagatc cttttaaattaaaaatgaag ttttaaatca atctaaagta 5760 tatatgagta aacttggtct gacagttaccaatgcttaat cagtgaggca cctatctcag 5820 cgatctgtct atttcgttca tccatagttgcctgactccc cgtcgtgtag ataactacga 5880 tacgggaggg cttaccatct ggccccagtgctgcaatgat accgcgagac ccacgctcac 5940 cggctccaga tttatcagca ataaaccagccagccggaag ggccgagcgc agaagtggtc 6000 ctgcaacttt atccgcctcc atccagtctattaattgttg ccgggaagct agagtaagta 6060 gttcgccagt taatagtttg cgcaacgttgttgccattgc tacaggcatc gtggtgtcac 6120 gctcgtcgtt tggtatggct tcattcagctccggttccca acgatcaagg cgagttacat 6180 gatcccccat gttgtgcaaa aaagcggttagctccttcgg tcctccgatc gttgtcagaa 6240 gtaagttggc cgcagtgtta tcactcatggttatggcagc actgcataat tctcttactg 6300 tcatgccatc cgtaagatgc ttttctgtgactggtgagta ctcaaccaag tcattctgag 6360 aatagtgtat gcggcgaccg agttgctcttgcccggcgtc aatacgggat aataccgcgc 6420 cacatagcag aactttaaaa gtgctcatcattggaaaacg ttcttcgggg cgaaaactct 6480 caaggatctt accgctgttg agatccagttcgatgtaacc cactcgtgca cccaactgat 6540 cttcagcatc ttttactttc accagcgtttctgggtgagc aaaaacagga aggcaaaatg 6600 ccgcaaaaaa gggaataagg gcgacacggaaatgttgaat actcatactc ttcctttttc 6660 aatattattg aagcatttat cagggttattgtctcatgag cggatacata tttgaatgta 6720 tttagaaaaa taaacaaata ggggttccgcgcacatttcc ccgaaaagtg ccacctgacg 6780 tctaagaaac cattattatc atgacattaacctataaaaa taggcgtatc acgaggccct 6840 ttcgtctcgc gcgtttcggt gatgacggtgaaaacctctg acacatgcag ctcccggaga 6900 cggtcacagc ttgtctgtaa gcggatgccgggagcagaca agcccgtcag ggcgcgtcag 6960 cgggtgttgg cgggtgtcgg ggctggcttaactatgcggc atcagagcag attgtactga 7020 gagtgcacca taaaattgta aacgttaatattttgttaaa attcgcgtta aatttttgtt 7080 aaatcagctc attttttaac caataggccgaaatcggcaa aatcccttat aaatcaaaag 7140 aatagcccga gatagggttg agtgttgttccagtttggaa caagagtcca ctattaaaga 7200 acgtggactc caacgtcaaa gggcgaaaaaccgtctatca gggcgatggc ccactacgtg 7260 aaccatcacc caaatcaagt tttttggggtcgaggtgccg taaagcacta aatcggaacc 7320 ctaaagggag cccccgattt agagcttgacggggaaagcc ggcgaacgtg gcgagaaagg 7380 aagggaagaa agcgaaagga gcgggcgctagggcgctggc aagtgtagcg gtcacgctgc 7440 gcgtaaccac cacacccgcc gcgcttaatgcgccgctaca gggcgcgtac tatggttgct 7500 ttgacgtatg cggtgtgaaa taccgcacagatgcgtaagg agaaaatacc gcatcaggcc 7560 gtaacctgtc ggatcaccgg aaaggacccgtaaagtgata atgattatca tctacatatc 7620 acaacgtgcg tggaggccat caaaccacgtcaaataatca attatgacgc aggtatcgta 7680 ttaattgatc tgcatcaact taacgtaaaaacaacttcag acaatacaaa tcagcgacac 7740 tgaatacggg gcaacctcat gtcaacgaagaacagaaccc gcagaacaac aacccgcaac 7800 atccgctttc ctaaccaaat gattgaacaaattaacatcg ctcttgagca aaaagggtcc 7860 gggaatttct cagcctgggt cattgaagcctgccgtcgga gactaacgtc agaaaagaga 7920 gcatatacat caattaaaag tgatgaagaatgaacatccc gcgttcttcc ctccgaacag 7980 gacgatattg taaattcact taattacgagggcattgcag taattgagtt gcagttttac 8040 cactttcctg acagtgacag actgcgtgttggctctgtca cagactaaat agtttgaatg 8100 attagcagtt atggtgatca gtcaaccaccagggaataat ccttcatatt attatcgtgc 8160 ttcaccaacg ctgcctcaat tgctctgaatgcttccagag acaccttatg ttctatacat 8220 gcaattacaa catcagggta actcatagaaatggtgctat taagcatatt ttttacacga 8280 atcagatcca cggagggatc atcagcagattgttctttat tcattttgtc gctccatgcg 8340 cttgctcttc atctagcggt taaaatattacttcaaatct ttctgtatga agatttgagc 8400 acgttggcct tacatacatc tgtcggttgtatttccctcc agaatgccag caggaccgca 8460 ctttgttacg caaccaatac tattaagtgaaaacattcct aatatttgac ataaatcatc 8520 aacaaaacac aaggaggtca gaccagattgaaacgataaa aacgataatg caaactacgc 8580 gccctcgtat cacatggaag gttttaccaatggctcaggt tgccattttt aaagaaatat 8640 tcgatcaagt gcgaaaagat ttagactgtgaattgtttta ttctgaacta aaacgtcaca 8700 acgtctcaca ttatatttac tatctagccacagataatat tcacatcgtg ttagaaaacg 8760 ataacaccgt gttaataaaa ggacttaaaaaggttgtaaa tgttaaattc tcaagaaaca 8820 cgcatcttat agaaacgtcc tatgataggttgaaatcaag agaaatcaca tttcagcaat 8880 acagggaaaa tcttgctaaa gcaggagttttccgatgggt tacaaatatc catgaacata 8940 aaagatatta ctataccttt gataattcattactatttac tgagagcatt cagaacacta 9000 cacaaatctt tccacgctaa atcataacgtccggtttctt ccgtgtcagc accggggcgt 9060 tggcataatg caatacgtgt acgcgctaaaccctgtgtgc atcgttttaa ttattcccgg 9120 acactcccgc agagaagttc cccgtcagggctgtggacat agttaatccg ggaatacaat 9180 gacgattcat cgcacctgac atacattaataaatattaac aatatgaaat ttcaactcat 9240 tgtttagggt ttgtttaatt ttctacacatacgattctgc gaacttcaaa aagcatcggg 9300 aataacacca tgaaaaaaat gctactcgctactgcgctgg ccctgcttat tacaggatgt 9360 gctcaacaga cgtttactgt tcaaaacaaaccggcagcag tagcaccaaa ggaaaccatc 9420 acccatcatt tcttcgtttc tggaattgggcagaagaaaa ctgtcgatgc agccaaaatt 9480 tgtggcggcg cagaaaatgt tgttaaaacagaaacccagc aaacattcgt aaatggattg 9540 ctcggtttta ttactttagg catttatactccgctggaag cgcgtgtgta ttgctcacaa 9600 taattgcatg agttgcccat cgcgatatgggcaactctat ctgcactgct cattaatata 9660 cttctgggtt ccttccagtt gtttttgcatagtgatcagc ctctctctga gggtgaaata 9720 atcccgttca gcggtgtctg ccagtcggggggaggctgca ttatccacgc cggaggcggt 9780 ggtggcttca cgcactgact gacagactgctttgatgtgc aaccgacgac gaccagcggc 9840 aacatcatca cgcagagcat cattttcagctttagcatca gctaactcct tcgtgtattt 9900 tgcatcgagc gcagcaacat cacgctgacgcatctgcatg tcagtaattg ccgcgttcgc 9960 cagcttcagt tctctggcat ttttgtcgcgctgggctttg taggtaatgg cgttatcacg 10020 gtaatgatta acagcccatg acaggcagacgatgatgcag ataaccagag cggagataat 10080 cgcggtgact ctgctcatac atcaatctctctgaccgttc cgcccgcttc tttgaatttt 10140 gcaatcaggc tgtcagcctt atgctcgaactgaccataac cagcgcccgg cagtgaagcc 10200 cagatattgc tgcaacggtc gattgcctgacggatatcac cacgatcaat cataggtaaa 10260 gcgccacgct ccttaatctg ctgcaatgccacagcgtcct gacttttcgg agagaagtct 10320 ttcaggccaa gctgcttgcg gtaggcatcccaccaacggg aaagaagctg gtagcgtccg 10380 gcgcctgttg atttgagttt tgggtttagcgtgacaagtt tgcgagggtg atcggagtaa 10440 tcagtaaata gctctccgcc tacaatgacgtcataaccat gatttctggt tttctgacgt 10500 ccgttatcag ttccctccga ccacgccagcatatcgagga acgccttacg ttgattattg 10560 atttctacca tcttctactc cggcttttttagcagcgaag cgtttgataa gcgaaccaat 10620 cgagtcagta ccgatgtagc cgataaacacgctcgttata taagcgagat tgctacttag 10680 tccggcgaag tcgagaaggt cacgaatgaaccaggcgata atggcgcaca tcgttgcgtc 10740 gattactgtt tttgtaaacg caccgccattatatctgccg cgaaggtacg ccattgcaaa 10800 cgcaaggatt gccccgatgc cttgttcctttgccgcgaga atggcggcca acaggtcatg 10860 tttttctggc atcttcatgt cttacccccaataaggggat ttgctctatt taattaggaa 10920 taaggtcgat tactgataga acaaatccaggctactgtgt ttagtaatca gatttgttcg 10980 tgaccgatat gcacgggcaa aacggcaggaggttgttagc gcgacctcct gccacccgct 11040 ttcacgaagg tcatgtgtaa aaggccgcagcgtaactatt actaatgaat tcaggacaga 11100 cagtggctac ggctcagttt gggttgtgctgttgctgggc ggcgatgacg cctgtacgca 11160 tttggtgatc cggttctgct tccggtattcgcttaattca gcacaacgga aagagcactg 11220 gctaaccagg ctcgccgact cttcacgattatcgactcaa tgctcttacc tgttgtgcag 11280 atataaaaaa tcccgaaacc gttatgcaggctctaactat tacctgcgaa ctgtttcggg 11340 attgcatttt gcagacctct ctgcctgcgatggttggagt tccagacgat acgtcgaagt 11400 gaccaactag gcggaatcgg tagtaagcgccgcctctttt catctcacta ccacaacgag 11460 cgaattaacc catcgttgag tcaaatttacccaattttat tcaataagtc aatatcatgc 11520 cgttaatatg ttgccatccg tggcaatcatgctgctaacg tgtgaccgca ttcaaaatgt 11580 tgtctgcgat tgactcttct ttgtggcattgcaccaccag agcgtcatac agcggcttaa 11640 cagtgcgtga ccaggtgggt tgggtaaggtttgggattag catcgtcaca gcgcgatatg 11700 ctgcgcttgc tggcatcctt gaatagccgacgcctttgca tcttccgcac tctttctcga 11760 caactctccc ccacagctct gttttggcaatatcaaccgc acggcctgta ccatggcaat 11820 ctctgcatct tgcccccggc gtcgcggcactacggcaata atccgcataa gcgaatgttg 11880 cgagcacttg cagtaccttt gccttagtatttccttcaag ctgcccctgc agg 11933 14 4999 DNA Artificial Sequence Vectorconstruct 14 cgcccctgca ggcagctgcg cgctcgctcg ctcactgagg ccgcccgggcaaagcccggg 60 cgtcgggcga cctttggtcg cccggcctca gtgagcgagc gagcgcgcagagagggagtg 120 gccaactcca tcactagggg ttcctgcggc cgcacgcgtg gtggcgcggggtaaactggg 180 aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaaccgtatataa 240 gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccccgcggcaggtaa 300 gtgccaggga atgtttgttc ttaaatacca tcgctccagg gaatgtttgttcttaaatac 360 catctactga cactgacatc cactttttct ttttctccac aggtatcgatccaccatgca 420 aatagagctc tccacctgct tctttctgtg ccttttgcga ttctgctttagtgccaccag 480 aagatactac ctgggtgcag tggaactgtc atgggactat atgcaaagtgatctcggtga 540 gctgcctgtg gacgcaagat ttcctcctag agtgccaaaa tcttttccattcaacacctc 600 agtcgtgtac aaaaagactc tgtttgtaga attcacggat caccttttcaacatcgctaa 660 gccaaggcca ccctggatgg gtctgctagg tcctaccatc caggctgaggtttatgatac 720 agtggtcatt acacttaaga acatggcttc ccatcctgtc agtcttcatgctgttggtgt 780 atcctactgg aaagcttctg agggagctga atatgatgat cagaccagtcaaagggagaa 840 agaagatgat aaagtcttcc ctggtggaag ccatacatat gtctggcaggtcctgaaaga 900 gaatggtcca atggcctctg acccactgtg ccttacctac tcatatctttctcatgtgga 960 cctggtaaaa gacttgaatt caggcctcat tggagcccta ctagtatgtagagaagggag 1020 tctggccaag gaaaagacac agaccttgca caaatttata ctactttttgctgtatttga 1080 tgaagggaaa agttggcact cagaaacaaa gaactccttg atgcaggatagggatgctgc 1140 atctgctcgg gcctggccta aaatgcacac agtcaatggt tatgtaaacaggtctctgcc 1200 aggtctgatt ggatgccaca ggaaatcagt ctattggcat gtgattggaatgggcaccac 1260 tcctgaagtg cactcaatat tcctcgaagg tcacacattt cttgtgaggaaccatcgcca 1320 ggcgtccttg gaaatctcgc caataacttt ccttactgct caaacactcttgatggacct 1380 tggacagttt ctactgtttt gtcatatctc ttcccaccaa catgatggcatggaagctta 1440 tgtcaaagta gacagctgtc cagaggaacc ccaactacga atgaaaaataatgaagaagc 1500 ggaagactat gatgatgatc ttactgattc tgaaatggat gtggtcaggtttgatgatga 1560 caactctcct tcctttatcc aaattcgctc agttgccaag aagcatcctaaaacttgggt 1620 acattacatt gctgctgaag aggaggactg ggactatgct cccttagtcctcgcccccga 1680 tgacagaagt tataaaagtc aatatttgaa caatggccct cagcggattggtaggaagta 1740 caaaaaagtc cgatttatgg catacacaga tgaaaccttt aagactcgtgaagctattca 1800 gcatgaatca ggaatcttgg gacctttact ttatggggaa gttggagacacactgttgat 1860 tatatttaag aatcaagcaa gcagaccata taacatctac cctcacggaatcactgatgt 1920 ccgtcctttg tattcaagga gattaccaaa aggtgtaaaa catttgaaggattttccaat 1980 tctgccagga gaaatattca aatataaatg gacagtgact gtagaagatgggccaactaa 2040 atcagatcct cggtgcctga cccgctatta ctctagtttc gttaatatggagagagatct 2100 agcttcagga ctcattggcc ctctcctcat ctgctacaaa gaatctgtagatcaaagagg 2160 aaaccagata atgtcagaca agaggaatgt catcctgttt tctgtatttgatgagaaccg 2220 aagctggtac ctcacagaga atatacaacg ctttctcccc aatccagctggagtgcagct 2280 tgaggatcca gagttccaag cctccaacat catgcacagc atcaatggctatgtttttga 2340 tagtttgcag ttgtcagttt gtttgcatga ggtggcatac tggtacattctaagcattgg 2400 agcacagact gacttccttt ctgtcttctt ctctggatat accttcaaacacaaaatggt 2460 ctatgaagac acactcaccc tattcccatt ctcaggagaa actgtcttcatgtcgatgga 2520 aaacccaggt ctatggattc tggggtgcca caactcagac tttcggaacagaggcatgac 2580 cgccttactg aaggtttcta gttgtgacaa gaacactggt gattattacgaggacagtta 2640 tgaagatatt tcagcatact tgctgagtaa aaacaatgcc attgaaccaagaagcttctc 2700 ccagaatcca ccagtcttga aacgccatca acgcgaaata actcgtactactcttcagtc 2760 agatcaagag gaaattgact atgatgatac catatcagtt gaaatgaagaaggaagattt 2820 tgacatttat gatgaggatg aaaatcagag cccccgcagc tttcaaaagaaaacacgaca 2880 ctattttatt gctgcagtgg agaggctctg ggattatggg atgagtagctccccacatgt 2940 tctaagaaac agggctcaga gtggcagtgt ccctcagttc aagaaagttgttttccagga 3000 atttactgat ggctccttta ctcagccctt ataccgtgga gaactaaatgaacatttggg 3060 actcctgggg ccatatataa gagcagaagt tgaagataat atcatggtaactttcagaaa 3120 tcaggcctct cgtccctatt ccttctattc tagccttatt tcttatgaggaagatcagag 3180 gcaaggagca gaacctagaa aaaactttgt caagcctaat gaaaccaaaacttacttttg 3240 gaaagtgcaa catcatatgg cacccactaa agatgagttt gactgcaaagcctgggctta 3300 tttctctgat gttgacctgg aaaaagatgt gcactcaggc ctgattggaccccttctggt 3360 ctgccacact aacacactga accctgctca tgggagacaa gtgacagtacaggaatttgc 3420 tctgtttttc accatctttg atgagaccaa aagctggtac ttcactgaaaatatggaaag 3480 aaactgcagg gctccctgca atatccagat ggaagatccc acttttaaagagaattatcg 3540 cttccatgca atcaatggct acataatgga tacactacct ggcttagtaatggctcagga 3600 tcaaaggatt cgatggtatc tgctcagcat gggcagcaat gaaaacatccattctattca 3660 tttcagtgga catgtgttca ctgtacgaaa aaaagaggag tataaaatggcactgtacaa 3720 tctctatcca ggtgtttttg agacagtgga aatgttacca tccaaagctggaatttggcg 3780 ggtggaatgc cttattggcg agcatctaca tgctgggatg agcacactttttctggtgta 3840 cagcaataag tgtcagactc ccctgggaat ggcttctgga cacattagagattttcagat 3900 tacagcttca ggacaatatg gacagtgggc cccaaagctg gccagacttcattattccgg 3960 atcaatcaat gcctggagca ccaaggagcc cttttcttgg atcaaggtggatctgttggc 4020 accaatgatt attcacggca tcaagaccca gggtgcccgt cagaagttctccagcctcta 4080 catctctcag tttatcatca tgtatagtct tgatgggaag aagtggcagacttatcgagg 4140 aaattccact ggaaccttaa tggtcttctt tggcaatgtg gattcatctgggataaaaca 4200 caatattttt aaccctccaa ttattgctcg atacatccgt ttgcacccaactcattatag 4260 cattcgcagc actcttcgca tggagttgat gggctgtgat ttaaatagttgcagcatgcc 4320 attgggaatg gagagtaaag caatatcaga tgcacagatt actgcttcatcctactttac 4380 caatatgttt gccacctggt ctccttcaaa agctcgactt cacctccaagggaggagtaa 4440 tgcctggaga cctcaggtga ataatccaaa agagtggctg caagtggacttccagaagac 4500 aatgaaagtc acaggagtaa ctactcaggg agtaaaatct ctgcttaccagcatgtatgt 4560 gaaggagttc ctcatctcca gcagtcaaga tggccatcag tggactctcttttttcagaa 4620 tggcaaagta aaggtttttc agggaaatca agactccttc acacctgtggtgaactctct 4680 agacccaccg ttactgactc gctaccttcg aattcacccc cagagttgggtgcaccagat 4740 tgccctgagg atggaggttc tgggctgcga ggcacaggac ctctactgactcgagcctaa 4800 taaaggaaat ttattttcat tgcaatagtg tgttggtttt ttgtgtgcggccgcaggaac 4860 ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcactgaggccgggc 4920 gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagcgagcgagcgc 4980 gcagctgcct gcaggacat 4999 15 14 PRT Artificial SequenceFactor VIII protein 15 Ser Phe Ser Gln Asn Pro Pro Val Leu Lys Arg HisGln Arg 1 5 10

What is claimed is:
 1. A method of treating hemophilia in a mammal,comprising: providing a pharmaceutical composition comprisingrecombinant adeno-associated virus virions, said virions comprising anucleotide sequence encoding a Factor VIII protein lacking at least aportion of the B domain, said nucleotide sequence operably linked toexpression control elements; and administering said pharmaceuticalcomposition to a mammal under conditions that result in the expressionof the Factor VIII protein at a level that provides a therapeutic effectin said mammal.
 2. The method of claim 9, wherein said Factor VIIIprotein is expressed in the liver.
 3. The method of claim 9, whereinsaid recombinant adeno-associated virus virions are administered to theliver.
 4. The method of claim 9, wherein said expression controlelements comprise a tissue-specific promoter.
 5. The method of claim 12wherein said expression control elements comprise a liver-specificpromoter.
 6. The method of claim 9 wherein said expression controlelements comprise a human growth hormone polyadenylation sequence. 7.The method of claim 9, wherein said recombinant adeno-associated virusvirions are administered via intravenous administration.
 8. The methodof claim 15, wherein said intravenous administration is via the portalvein.
 9. The method of claim 9, wherein said recombinantadeno-associated virus virions are administered via intraarterialadministration.
 10. The method of claim 17, wherein said recombinantadeno-associated virus virions are administered via the hepatic artery.11. The method of claim 9, wherein said nucleotide sequence encodingFactor VIII comprises a light chain and a heavy chain and wherein saidlight chain and heavy chain are operably linked by a junction.
 12. Themethod of claim 19, wherein said nucleotide sequence is SEQ ID 13, suchthat said junction has the amino acid sequence Ser-Phe.
 13. The methodof claim 19, wherein said nucleotide sequence is SEQ ID 14, such thatsaid junction has the amino acid sequenceSer-Phe-Ser-Gln-Asn-Pro-Pro-Val-Leu-Lys-Arg-His-Gln-Arg.
 14. The methodof claim 19, wherein said expression control elements comprise aliver-specific promoter, and wherein said recombinant adeno-associatedvirus virions are administered to the liver of said mammal.